Composition of Novel Carbohydrate Drug for Treatment of Human Diseases

ABSTRACT

Aspects of the invention provide compositions for use in the treatment galectin-dependent diseases. In particular, compositions comprising a selectively depolymerized, branched galactoarabino-rhamnogalacturonate whose backbone is predominantly comprised of 1,4-linked galacturonic acid (GalA) moieties, with a lesser backbone composition of alternating 1,4-linked GalA and 1,2-linked rhamnose (Rha), which in-turn is linked to any number of side chains, including predominantly 1,4-b-D-galactose (Gal) and 1,5-a-L-arabinose (Ara) residues.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/525,972, filed Jul. 30, 2019, which is a continuation of U.S.application Ser. No. 15/973,716, filed May 8, 2018, now U.S. Pat. No.10,420,793, which is a continuation of U.S. application Ser. No.15/478,599, filed Apr. 4, 2017, now U.S. Pat. No. 9,974,802, which is acontinuation of U.S. application Ser. No. 14/575,062, filed Dec. 18,2014, now U.S. Pat. No. 9,649,327, which is a continuation of U.S.application Ser. No. 14/456,644, filed Aug. 11, 2014, now U.S. Pat. No.8,962,824, which is a continuation of U.S. application Ser. No.13/573,442, filed Sep. 14, 2012, now U.S. Pat. No. 8,871,925, whichclaims the benefit of and priority to U.S. provisional Application Ser.No. 61/580,830, filed Dec. 28, 2011, the entire disclosures of each ofwhich are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

Aspects of the invention relate to a pharmaceutical-gradepolysaccharide, pharmaceutical compositions thereof and to methods ofmanufacturing the same. Other aspects of the invention relate to methodsof treating disease conditions, such as medical conditions related toinflammation and fibrosis and cancer linked at least in part to aberrantor increased expression of galectin proteins, using these compounds andcompositions.

BACKGROUND OF THE INVENTION

Plants and plant products have been used for many years to derivepharmaceutical products, often in the form of specific complex organicmolecules that have physiological function in animals and human. Inaddition to single specific molecules, the structural components of theplant cell, which are large, complex carbohydrate molecules, have beenexplored for various effects on animals and humans in normal physiologyand disease. Among the complex carbohydrates that make up the cell wallsof plant cells, pectins represent a class of molecules that have beenextensively examined.

In order to evaluate the effect of pectins in the systemic circulationand organs of animals and humans, attempts have been made to developmodified pectins that could be utilized as medicinal compounds.

Accordingly, there is a need to provide modified pectins and method ofmanufacturing modified pectins for use as parenteral or enteralmedicinal compounds. Moreover, there is a need to for these compounds tohave the required pharmacological properties to inhibit inflammation andfibrosis while remaining non-toxic to other cells and tissues.

SUMMARY OF THE INVENTION

Aspects of the invention relate to a compound or composition comprisinga compound in an acceptable pharmaceutical carrier for parenteral orenteral administration, for use in therapeutic formulations.

In some embodiments, the compound is a polysaccharide and may bechemically defined as arabinogalacto-rhamnogalacturonan (herein referredto as Compound G), a selectively depolymerized, branched heteropolymerwhose backbone is predominantly comprised of 1,4-linked galacturonicacid (GalA) and methyl galacturonate (MeGalA) residues, with a lesserbackbone composition of alternating 1,4-linked GalA and 1,2-linkedrhamnose (Rha), which in-turn is linked to any number of oligomer sidechains, including predominantly 1,4-β-D-galactose (Gal),1,5-α-L-arabinose (Ara) residues or combination thereof. Other sidechain minor constituents may include xylose (Xyl), glucose (Glu), fucose(Fuc) or any combination of the foregoing.

In some embodiments, the arabinogalacto-rhamnogalacturonan compound ofthe present invention (Compound G) is capable of reducing the secretionof TNF-alpha cytokine from monocytes stressed with endotoxin. In someembodiments, the compound G is capable of reducing the secretion of TNFalpha by activated macrophages by at least 25%.

In some embodiments, the 1,4-linked galacturonic acid and methylgalacturonate residues backbone can represent between 55 to 85 molarpercent of the total carbohydrate molar content, the branchedheteropolymer of alternating α-1,2 linked rhamnose and α-1,4-linked GalAresidues can represent between 1 and 6 molar percent of the totalcarbohydrate molar content, the oligomer 1,4-β-D-galactose of theprimary branching can represent between 6 to 15 molar percent of thetotal carbohydrate molar content and the oligomer 1,5-α-L-arabinose ofthe primary branching can represent between 2 to 8 molar percent of thetotal carbohydrate molar content, as characterized by gaschromatography/mass spectrometry.

In some embodiments, the oligomer of 1,4-β-D-galactose residues,1,5-α-L-arabinose residues or combinations thereof represents at least 8molar percent of the total carbohydrate molar content.

In some embodiments, the 1,4-β-D-galactose and 1,5-α-L-arabinoseresidues are present in a 2:1 to a 3:1 ratio in thearabinogalacto-rhamnogalacturonan compound of the present invention.

In some embodiments, the compound of the present invention has a degreeof methoxylation ranging from 40% to 70% of the maximum of 87%.

In some embodiments, the compound of the present invention has a methylgalacturonate to galacturonic acid ratio ranging from 2:1 to 1:2.

In some embodiments, the compound of the present invention has a methylgalacturonate plus galacturonic acid ratio to galactose ranging from 4:1to 7:1.

In some embodiments, the molar percent of the 1,4-β-D-Gal and1,5-α-L-Ara residues in the compound of the present invention can exceed10% of the total molar carbohydrates with approximate ratio ranging from1:1 to 3:1 respectively.

In some embodiments, the compound is a polysaccharide chemically definedas galactoarabino-rhamnogalacturonate (Compound G), a branchedheteropolymer with average molecular weight distribution of 2,000 to80,000, or 20,000 to 70,000, or 5,000 to 55,000 Daltons, as determinedby SEC-RI and/or the SEC-MALLS method.

In some embodiments, the compound can be a highly soluble modifiedpolysaccharide sufficiently reduced in average molecular weight, forexample from about 2,000 to about 80,000 D, so as to be compatible withtherapeutic formulations for pluralistic administration via routesincluding but not limited to intravenous, subcutaneous, intra-articular,inhaled, and oral.

In some embodiments, the compound can be substantially free of microbialendotoxin, agricultural pesticides, agricultural herbicides, copper,heavy metals, proteins, nitrogenous compounds or any combination of theforegoing.

In some embodiments, the compound can be synthesized from natural,highly branched, minimally processed and high methoxylated USP pectinwhich may come from any plant source, including but not limited to,citrus fruits, apple, or beet.

In some embodiments, the compound can be synthesized from natural,highly branched, minimally processed and high methoxylated USP pectinlike one manufactured from apple pomace containing 8-12% pectin.

In some embodiments, the compound can be synthesized under asufficiently controlled and specific hydrolysis of the glycosidic-linkedmethoxylated α-1,4-linked GalA while preserving the side-chains withenriched amounts of 1,4-β-D-Gal and 1,5-α-L-Ara. Amounts of 1,4-β-D-Galand 1,5-α-L-Ara can be quantitatively determined by GC-MS (Gaschromatography-mass spectroscopy) and AELC-PAD (anion exchange liquidchromatography-pulsed amperometric detector) methods.

In some embodiments the compound can be produced by a process comprisingdepolymerization catabolized by targeted peroxidation cleavage ofglycosidic bonds by ionized OH sup-generated from ascorbic acid and/orperoxide in presence or absence of additional reduced form of atransition metal ion, like Cu sup.++. at 1 to 100 mM. Other transitionmetals like Ca. sup.++ or Fe.sup.++ can also be used for this purpose.

In some embodiments, the depolymerized compound can be exposed to pH ofbetween 8 to10 for 10 to 30 minutes at temperature of 2 to 60° C. toinitiate controlled limited demethoxylation to generate a depolymerizedcompound with a degree of methoxylation of 40 to 70 percent incomparison to initial levels of maximum 87% and can be referred to asmiddle-methoxylated compound. Complete methoxylation of galacturonicacid is considered to be approximately DE 87%.

In some embodiments, the depolymerized composition can be exposed tomultiple washes of hot acidic alcohol (e.g at temperatures ranging from30 to 80° C.) to remove any residual endotoxin, copper and heavy metals,agricultural contaminates and other impurities.

In some embodiments, the compound does not induce decreased viabilitywhen used to treat LX2 immortalized human hepatic stellate cells.

In some embodiments, the compound is capable of reducing expression ofgalectin-3 at the cell surface or substantial decrease in secretion ofgalectin-3 in the media when used to treat stressed LX2 immortalizedhuman hepatic stellate cells producing galectin-3.

Aspects of the invention relate to an arabinogalacto-rhamnogalcturonancompound comprising a 1,4-linked galacturonic acid (GalA) and methylgalacturonate (MeGalA) residues backbone linked to branchedheteropolymers of alternating oligomers of α-1,2 linked rhamnose andα-1,4-linked GalA residues, the rhamnose residues carrying a primarybranching of oligomers of 1,4-β-D-galactose residues, 1,5-α-L-arabinoseresidues, or combinations thereof, wherein the compound is capable ofreducing the secretion of TNF alpha cytokine from monocytes stressedwith endotoxin, wherein the compound does not inhibit cancer cellproliferation in a cancer cell apoptosis or a cytotoxic model, andwherein the compound is not cytotoxic to monocytes or activatedmonocytes .

In some embodiments, the compound does not inhibit cancer cellproliferation in a cancer cell and is not cytotoxic to monocytes oractivated monocytes at concentrations up to 500 μg/mL.

In some embodiments, the 1,4-linked galacturonic acid and the methylgalacturonate residues backbone can represent between 55 to 85 molarpercent of the total carbohydrate molar content, the branchedheteropolymer of alternating α-1,2 linked rhamnose and α-1,4-linked GalAresidues can represent between 1 and 3 molar percent of the totalcarbohydrate molar content, the oligomer 1,4-β-D-galactose of theprimary branching can represent between 6 to 15 molar percent of thetotal carbohydrate molar content and the oligomer 1,5-α-L-arabinose ofthe primary branching can represent between 2 to 8 molar percent of thetotal carbohydrate molar content, as characterized by gaschromatography/mass spectrometry.

In some embodiments, the 1,4-β-D-galactose residues, 1,5-α-L-arabinoseresidues or combination thereof can represent at least 8 molar percentof the total carbohydrate molar content.

In some embodiments, the 1,4-β-D-galactose and 1,5-α-L-arabinoseresidues can be present in a 2:1 ratio.

In some embodiments, the compound can have an average molecular weightranging from 5 kDa to 55 kDa or ranging from 2 kDa to 70 kDa.

In some embodiments, the compound can have a degree of methoxylationranging from 40% to 70% out of maximum 87%.

In some embodiments, the compound can have a methyl galacturonate togalacturonic acid ratio ranging from 2:1 to 1:2.

In some embodiments, the compound can have a methyl galacturonate plusgalacturonic acid ratio to galactose ranging from 4:1 to 8:1.

In some embodiments, the compound does not induce decreased viabilitywhen used to treat LX2 immortalized human hepatic stellate cells.

In some embodiments, the compound is capable of inducing substantialdecrease in expression of galectin-3 at the cell surface or substantialdecrease in secretion of galectin-3 in the media when used to treatstressed LX2 immortalized human hepatic stellate cells producinggalectin-3.

Aspects of the invention relate to compositions comprising a compound inan acceptable pharmaceutical carrier for use in therapeuticformulations. In some embodiments, the composition can be administeredparenterally via an intravenous, subcutaneous, or oral route.

In some embodiments, the composition can further comprise a therapeuticagent. For example, the therapeutic agent can be an anti-oxidantcompound, an anti-inflammatory agent, vitamins, a neutraceuticalsupplement or combinations thereof.

In some embodiments, the composition can be used in the treatment ofnonalcoholic steatohepatitis, fibrosis, inflammatory and autoimmunedisorders, neoplastic conditions or of cancer.

In some embodiments, the composition can be used in the treatment ofliver fibrosis, kidney fibrosis, lung fibrosis, or heart fibrosis.

In some embodiments, the invention relates to a composition or acompound utilized in treating or a method of treating inflammatory andfibrotic disorders in which galectins are at least in part involved inthe pathogenesis, including but not limited to enhanced anti-fibrosisactivity in organs, including but not limited to liver, kidney, lung,and heart.

In some embodiments, the invention relates to a composition or acompound that has therapeutic activity or a method to reduce thepathology and disease activity associated with nonalcoholicsteatohepatitis (NASH) including but not limited to steatosis (fataccumulation in hepatocytes), ballooning degeneration of hepatocytes,inflammatory infiltrate in the liver, and deposition of collagen orfibrosis.

In some embodiments, the invention relates to a composition or acompound utilized in treating or a method of treating inflammatory andautoimmune disorders in which galectins are at least in part involved inthe pathogenesis including but not limited to arthritis, rheumatoidarthritis, asthma, and inflammatory bowel disease.

In some embodiments, the invention relates to a composition or acompound utilized in treating or a method of treating neoplasticconditions (e.g. cancers) in which galectins are at least in partinvolved in the pathogenesis by inhibiting processes promoted by theincrease in galectins, including but not limited to tumor cell invasion,metastasis, and neovascularization.

In some embodiments, the invention relates to a composition or acompound utilized in enhancing or a method for enhancing the ability oftumor infiltrating T-cells, which are inhibited at least in part by theeffect of tumor derived galectin proteins, to more effectively identifyand kill tumor cells and thereby slow, stop or reverse the progressionof tumors.

In some embodiments, a therapeutically effective amount of thedepolymerized compound or of the composition can be compatible andeffective in combination with a therapeutically effective amount ofvarious anti-inflammatory drugs, vitamins, other pharmaceuticals andnutraceuticals drugs, without limitation.

In some embodiments, a therapeutically effective amount of thedepolymerized compound or of the composition can be compatible andeffective in combination with a therapeutically effective amount ofvarious anti-oxidant compounds such as glycyrrhizin, ascorbic acid,L-glutathione, cysteamine and the like or combinations thereof.

In some embodiments, a therapeutically effective amount of the compoundor of the composition can be non-toxic and does not induce apoptosis incultured cell lines when added to cell culture media including but notlimited to the cell lines LX-2, or other stellate cells.

In some embodiments, a therapeutically effective amount of the compoundor of the composition is not cytotoxic to mammalian cultured cells whenadded to cell culture media including but not limited to B16-F10melanoma cells, THP-1 monocyte/macrophage cells, primary peripheralblood mononuclear cells (PBMC) and MRC-5 lung fibroblast cells.

In some embodiments, a therapeutically effective amount of the compoundor of the composition can have an anti-inflammatory effect on cell linesas measured by production of pro-inflammatory cytokines including butnot limited to TNF-alpha.

In some embodiments, efficacy of the compound or of the composition fortreatment of liver fibrosis can be determined by administering thecompound or composition to animal models of fibrosis including but notlimited to rats injected intraperitoneally with the chemical toxinthioacetamide, resulting in at least 5% to 25% reduction in livercollagen content as determined by morphometric quantification.

In another embodiment, efficacy of the composition for treatment of NASHcan be determined by administering the compound or composition to animalmodels of NASH including but not limited to mice rendered diabetic andfed a high fat diet, resulting in at least 5% reduction inhepatocellular fat accumulation, at least a 5% reduction in the numberof hepatocytes with ballooning degeneration, at least 5% reduction inliver infiltration of inflammatory cells, and at least a 5% reduction inliver collagen content as determined by morphometric quantification(assessed by staining positive for Sirius red).

In another embodiment, administering a therapeutically effective amountof the depolymerized compound or of the composition to an animal modelof NASH can result in reduction of fibrosis as measured by standardhistopathology of biopsies, reduction in disease activity by NAFLDgrading, a decrease in the number of cells expressing alpha SmoothMuscle Actin, or a decrease in other inflammatory mediators or adecrease in lipid trafficking and metabolism enzymes including but notlimited to CD36.

In another embodiment, administering a therapeutically effective amountof the depolymerized compound or of the composition can result inreduction of galectin-3 as measured by either level of mRNA or theexpression of the galactose binding protein.

In another embodiment, administering a therapeutically effective amountof the depolymerized compound or of the composition can result inreduced growth, invasion, metastasis, or increased sensitivity to theinnate immune system of syngeneic or xenotopic tumors in animals.

These and other aspects of the present invention will become apparentupon reference to the following detailed description.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The present invention will be further explained with reference to theattached drawings, wherein like structures are referred to by likenumerals throughout the several views. The drawings shown are notnecessarily to scale, with emphasis instead generally being placed uponillustrating the principles of the present invention.

FIG. 1 shows a schematic of a manufacturing process flow chart that maybe utilized in one embodiment to manufacture Compound G, the activepharmaceutical ingredient.

FIG. 2 shows an illustration of the molecular structure of Compound Gaccording to some embodiments.

FIG. 3a shows a Zimm plot calculation of average molecular weightcalculated for the selectively depolymerized compound of the instantinvention (Compound G) as determined by Multi-Angel Laser ScatteringAnalyzer (MALS) method. FIG. 3b shows a Zimm plot calculation of do/dcparameters calculated for the selectively depolymerized compound(Compound G) of the instant invention in a 10 nM EDTA buffered solutionas determined by Multi-Angel Laser Scattering Analyzer (MALS) method.

FIG. 4a shows the determination of molecular weight distribution of theselectively depolymerized compound (Compound G) of the instant inventionusing a size exclusion chromatography (SEC) method. The standard curveis established by reference to polysaccharides molecular weight (MW)standards monitored by Refractive Index (RI) detector which are used tocalculate the average molecular weight. FIG. 4b shows the profile of theselectively depolymerized compound (Compound G) of the instant inventionas characterized by Refractive Index (RI) detector.

FIG. 5a shows two dimensional NMR (2-D NMR) of the combined spectrum ofD1 and C13 NMR for Compound G. This two dimensional NMR depicts afinger-printing identification of the glycosidic linkages of Compound G.FIG. 5b shows two dimensional NMR (2-D NMR) of the combined spectrum ofD1 and C13 NMR for Compound S. FIG. 5c shows an overlap comparison ofthese spectra demonstrating the differences between Compound G andCompound S. The circled signals are specific to Compound G.

FIG. 6a-d is a depiction of various types of experiments demonstratingthe lack of cytotoxicity and apoptosis of Compound G on culturedstellate liver cell line LX-2. FIG. 6a is a graph showing theincorporation of radiolabeled thymidine in cultured LX-2 cells. FIG. 6bis a graph showing the evaluation of cellular viability in cultured LX-2cells. FIG. 6c depicts an assessment of apoptosis and cell cycle by FACSin cultured LX-2 cells. FIG. 6d shows an assessment of apoptosis by DNAfragmentation in cultured LX-2 cells using Annexin V apoptosis detectionkit APC (eBioscience).

FIG. 7 is a graph showing the relative cytotoxicity on themonocyte/macrophage cell line THP-1 following 3 days in the presence ofCompound G of the present invention (square), Compound D, agalactomannan product (diamond) and digitoxin (triangle).

FIG. 8 is a graph showing the relative cytotoxicity on cultured melanomacell line B16-F10 following 3 days in the presence of Compound G of thepresent invention (diamond), Compound D, a galactomannan product(square) and 5-Fluoruracil (triangle).

FIG. 9 is a graph showing the lack of cytotoxicity of Compound G oncultured peripheral blood mononuclear cells (PBMC), a primary cellculture.

FIG. 10 is a graph showing the lack of cytotoxicity on growth of lungfibroblast (MRC-5*) cells after 4 days growth in present of Compounds Gand Compound D. Cytotoxicity was measured using MTS assay.

FIG. 11 is a graph showing the relative anti-inflammatory effect ofthree modified polysaccharides including compound G of the currentinvention, on secretion of TNF-alpha by PBMC cells stressed withmicrobial endotoxin (50 ng/ml).

FIG. 12 is a comparison of Compound G of the present invention andCompound H on the secretion of TNF-alpha by PBMC cells stressed withmicrobial endotoxin (50 ng/ml).

FIG. 13 is a photograph of immunostaining of galectin-3 with MAb-HRPdepicting the anti-galectin suppression effect of Compound G onsecretion of galectin-3 by LX-2 cells.

FIG. 14 is a depiction of an experimental rat fibrosis model induced bythioacetamide injection (TAA model, a chemical toxicity liver fibrosismodel).

FIG. 15 is a photographic depiction of histological regression offibrosis (Sirius Red staining for collagen) after 4 weekly treatmentswith Compound G of TAA-induced liver fibrosis.

FIG. 16 is a graphical and statistical comparison of percentage ofcollagen in liver (as a measure of fibrosis) in the TAA model after 4weekly treatments with vehicle control, Compound D and Compound G.Sirius Red staining was used for quantitative measurement of percentcollagen in the histological slides.

FIG. 17 is a depiction of the mouse fatty-liver fibrosis model, a NASHexperimental model associated with metabolic disorder (severe diabetes)and high fat diet. Also shown is the experimental design of treatmentwith Compound G and Compound D.

FIG. 18 is a graph showing the change in body weight of NASH micetreated with vehicle, Compound D, or Compound G.

FIGS. 19A-B show the statistical significance of the extent of depictingthe extent of liver cell steatosis, ballooning degeneration, and lobularinflammation (as summarized in a NAFLD activity score) in mice treatedwith vehicle, Compound D, and Compound G. FIG. 19A shows the NAFLDactivity score in mice treated with vehicle, Compound D, and Compound Gfor 6 to 9 weeks (Early Rx). FIG. 19B shows the NAFLD activity score inmice treated with vehicle, Compound D, and Compound G for 9-12 weeks(Late Rx).

FIGS. 20A-B show the statistical significance of the extent of percentcollagen in liver sections as measured by the Sirius Red stainingmethod, for NASH mice treated with vehicle, Compound D, and Compound G.FIG. 20A shows the Sirius-Red positive area (%) in mice treated withvehicle, Compound D, and Compound G for 6 to 9 weeks (Early Rx). FIG.20B shows the Sirius-Red positive area (%) in mice treated with vehicle,Compound D, and Compound G for 9-12 weeks (Late Rx).

DETAILED DESCRIPTION OF THE INVENTION

Detailed embodiments of the present invention are disclosed herein;however, it is to be understood that the disclosed embodiments aremerely illustrative of the invention that may be embodied in variousforms. In addition, each of the examples given in connection with thevarious embodiments of the invention is intended to be illustrative, andnot restrictive. Further, the figures are not necessarily to scale, somefeatures may be exaggerated to show details of particular components. Inaddition, any measurements, specifications and the like shown in thefigures are intended to be illustrative, and not restrictive. Therefore,specific structural and functional details disclosed herein are not tobe interpreted as limiting, but merely as a representative basis forteaching one skilled in the art to variously employ the presentinvention.

Citation of documents herein is not intended as an admission that any ofthe documents cited herein is pertinent prior art, or an admission thatthe cited documents are considered material to the patentability of theclaims of the present application.

Unless otherwise specified, all percentages expressed herein areweight/weight.

Pectins are comprised of diversified large carbohydrate polymers thatare composed of a backbone of polymeric galacturonic acid with periodicinterspersed rhamnose molecules and periodic branched chains of variouscarbohydrates including galactose, arabinose, fucose, glucose, andothers. The galacturonic acid molecules are naturally modified bymethoxylation at a high percentage of the residues.

The large pectin carbohydrate polymers have been studied in their nativeform (directly after extraction from plants) and after various degreesof modification which hydrolyze and chemically modify the polymers.While modified pectins are widely used in the food industry for theirability to gel and provide consistency to various food preparations,they have also been evaluated as potential medicinal compounds.

Plant-derived pectin materials, both unaltered and modified, have beenextensively evaluated when administered orally to animals and humans. Avariety of effects of orally administered unaltered and modified pectinshave been described including increased satiety, weight loss,modification of bowel motility, and effects on bowel function includingsalutatory effects on constipation and diarrhea. Ingested pectinmaterial is not digested by normal gut enzymes nor absorbed by theintestine. Therefore, there is little to no pectin that is absorbed intothe blood stream after oral absorption.

In order to evaluate the effect of pectins and modified pectin moleculesin the systemic circulation and organs of animals and humans, attemptshave been made to develop modified pectins that could be utilized asparenteral compounds. Pectin derived from a mixture of citrus fruit hasbeen evaluated in cell culture models and in animal models of disease,particular in cancer cells and models of cancer in animals. The use ofmodified citrus pectin has been shown to cause cytotoxicity andapoptosis in animal cell lines. Such modified citrus pectin has beenproposed to be useful in malignant conditions (See U.S. Pat. No.8,128,966).

Galectins

Galectins (also known as galaptins or S-lectin) are a family of lectinswhich bind beta-galactoside. Galectin as general name was proposed in1994 for a family of animal lectins (Barondes, S. H., et al.: Galectins:a family of animal beta-galactoside-binding lectins. Cell 76, 597-598,1994), The family is defined by having at least one characteristiccarbohydrate recognition domain (CRD) with an affinity forbeta-galactosides and sharing certain sequence elements. Within the samepeptide chain, some galectins have a CRD with only a few additionalamino acids, whereas others have two CRDs joined by a link peptide, andone (galectin-3) has one CRD joined to a different type of domain. Thegalectin carbohydrate recognition domain (CRD) is a beta-sandwich ofabout 135 amino acids. The two sheets are slightly bent with 6 strandsforming the concave side and 5 strands forming the convex side. Theconcave side forms a groove in which carbohydrate is bound (Leffler H,Carlsson S, Hedlund M, Qian Y, Poirier F (2004). “Introduction togalectins”. Glycoconj. J. 19 (7-9): 433-40).

A wide variety of biological phenomena have been shown to be related togalectins, e.g., development, differentiation, morphogenesis, tumormetastasis, apoptosis, RNA splicing, etc. However, relatively little isknown about the mechanism by which galectins exert these functions,particularly in terms of carbohydrate recognition.

Generally, the carbohydrate domain binds to galactose residuesassociated with glycoproteins. At least fifteen mammalian galectinproteins have been identified which have one or two carbohydrate domainsin tandem.

Galectin proteins are found in the intracellular space where they havebeen assigned a number of functions and are secreted into theextracellular space. In the extracellular space, galectin proteins canhave multiple functions including promoting interactions betweenglycoproteins that may lead to reduced function, or enhanced functions,or in the case of integral membrane glycoprotein receptors, modificationof cellular signaling (Sato et al “Galectins as danger signals inhost-pathogen and host-tumor interactions: new members of the growinggroup of “Alarmins.” In “Galectins,” (Klyosov, et al eds.), John Wileyand Sons, 115-145, 2008, Liu et al “Galectins in acute and chronicinflammation,” Ann. N. Y. Acad. Sci. 1253: 80-91, 2012). Galectinproteins in the extracellular space can additionally promote cell-celland cell matrix interactions (Wang et al., “Nuclear and cytoplasmiclocalization of galectin-1 and galectin-3 and their roles in pre-mRNAsplicing.” In “Galectins” (Klyosov et al eds.), John Wiley and Sons,87-95, 2008).

Galectins have been shown to have domains which promotehomodimerization. Thus, galectins are capable of acting as a “molecularglue” of sorts between glycoproteins. Galectins are found in multiplecellular compartments, including the nucleus and cytoplasm, and aresecreted into the extracellular space where they interact with cellsurface and extracellular matrix glycoproteins. The mechanism ofmolecular interactions can depend on the localization. While galectinscan interact with glycoproteins in the extracellular space, theinteractions of galectin with other proteins in the intracellular spacegenerally occurs via protein domains. In the extracellular space theassociation of cell surface receptors may increase or decrease receptorsignaling or the ability to interact with ligands. Galectin proteins aremarkedly increased in a number of animal and human disease states,including but not limited to diseases associated with inflammation,fibrosis, autoimmunity, and neoplasia. Galectins have been directlyimplicated in the disease pathogenesis, as described below. For example,diseases states that may be dependent on galectins include, but are notlimited to, acute and chronic inflammation, allergic disorders, asthma,dermatitis, autoimmune disease, inflammatory and degenerative arthritis,immune-mediated neurological disease, fibrosis of multiple organs(including but not limited to liver, lung, kidney, pancreas, and heart),inflammatory bowel disease, atherosclerosis, heart failure, ocularinflammatory disease, a large variety of cancers.

In addition to disease states, galectins are important regulatorymolecules in modulating the response of immune cells to vaccination,exogenous pathogens and cancer cells.

One of skill in the art will appreciate that compounds that can bind togalectins and/or alter galectin's affinity for glycoproteins, reducehetero- or homo-typic interactions between galectins, or otherwise alterthe function, synthesis, or metabolism of galectin proteins may haveimportant therapeutic effects in galectin-dependent diseases.

Galectins show an affinity for galactose residues attached to otherorganic compounds, such as in lactose [(β-D-Galactosido)-D-glucose],N-acetyl-lactosamine, poly-N-acetyllactosamine, galactomannans,fragments of pectins, as well as other galactose containing compounds.It should be noted that galactose by itself does not bind to galectins,or binds so weakly that the binding can hardly be detected.

Pectin and modified pectin have been shown to bind to galectin proteinspresumably on the basis of containing galactose residues that arepresented in the context of a macromolecule, in this case a complexcarbohydrate rather than a glycoprotein in the case of animal cells.

Galectin proteins have been shown to be markedly increased ininflammation, fibrotic disorders, and neoplasia (Ito et al. “Galectin-1as a potent target for cancer therapy: role in the tumormicroenvironment”, Cancer Metastasis Rev. PMID: 22706847 (2012),Nangia-Makker et al. Galectin-3 binding and metastasis,” Methods MolBiol. 878: 251-266, 2012, Canesin et al. Galectin-3 expression isassociated with bladder cancer progression and clinical outcome,” TumourBiol. 31: 277-285, 2010, Wanninger et al. “Systemic and hepatic veingalectin-3 are increased in patients with alcoholic liver cirrhosis andnegatively correlate with liver function,” Cytokine. 55: 435-40, 2011.Moreover, experiments have shown that galectins, particularly galectin-1and galectin-3, are directly involved in the pathogenesis of theseclasses of disease (Toussaint et al., “Galectin-1, a gene preferentiallyexpressed at the tumor margin, promotes glioblastoma cell invasion.”,Mol Cancer. 11:32, 2012, Liu et al 2012, Newlaczyl et al., “Galectin-3—ajack-of-all-trades in cancer,” Cancer Lett. 313: 123-128, 2011, Banh etal., “Tumor galectin-1 mediates tumor growth and metastasis throughregulation of T-cell apoptosis,” Cancer Res. 71: 4423-31, 2011, Lefrancet al., “Galectin-1 mediated biochemical controls of melanoma and gliomaaggressive behavior,” World J. Biol. Chem. 2: 193-201, 2011, Forsman etal., “Galectin 3 aggravates joint inflammation and destruction inantigen-induced arthritis,” Arthritis Reum. 63: 445-454, 2011, de Boeret al., “Galectin-3 in cardiac remodeling and heart failure,” Curr.Heart Fail. Rep. 7, 1-8, 2010, Ueland et al., “Galectin-3 in heartfailure: high levels are associated with all-cause mortality,” Int JCardiol. 150: 361-364, 2011, Ohshima et al., “Galectin 3 and its bindingprotein in rheumatoid arthritis,” Arthritis Rheum. 48: 2788-2795, 2003).

Therefore, there is a need to identify therapeutics that have affectgalectins involved in human disorders, such as inflammatory diseases,fibrotic diseases, neoplastic diseases or combinations thereof, and thathave a reliable safety profile, so as to be used in therapeutics.

Chemically Modified Pectins and Compositions

Compositions comprising a chemically modified pectin derived from applepectin and methods of manufacturing such modified apple pectin having anactivity in a cellular assay of inflammatory fibrosis have beenpreviously described [see U.S. Pat. No. 8,236,780, incorporated hereinby reference in its entirety]. Such modified pectin has been shown toreduce the induction of liver derived fibrogenic cells while having noeffect on viability of the cells.

Aspects of the invention relate to a chemically modified pectin ormodified pectin composition and methods of producing a modified pectinor modified pectin composition having an anti-inflammatory and/oranti-fibrogenic effects. In some embodiments, the modified pectin is apolysaccharide chemically defined as galactoarabino-rhamnogalacturonate.

Compositions for parenteral or enteral administration to a subject aredisclosed herein. In some embodiments, the composition can comprise apectin derivative or modified pectin compound of the invention in anacceptable pharmaceutical carrier. The term “pharmaceutically acceptablecarrier” refers to a carrier or adjuvant that may be administered to asubject (e.g., a patient), together with a compound of this invention,and which does not destroy the pharmacological activity thereof and isnontoxic when administered in doses sufficient to deliver a therapeuticamount or an effective mount of the compound.

“Pharmaceutically acceptable carrier” refers to any and all solvents,dispersion media, e.g., human albumin or cross-linked gelatinpolypeptides, coatings, antibacterial and antifungal compounds,isotonic, e.g., sodium chloride or sodium glutamate, and absorptiondelaying compounds, and the like that are physiologically compatible.The use of such media and compounds for pharmaceutically activesubstances is well known in the art. Preferably, the carrier is suitablefor oral, intravenous, intramuscular, subcutaneous, parenteral, spinalor epidural administration (e.g., by injection or infusion). Dependingon the route of administration, the active compound can be coated in amaterial to protect the compound from the action of acids and othernatural conditions that can inactivate the compound.

In some embodiments, the modified pectin compound is obtained from apharmaceutical-grade pectin.

In some embodiments, the modified pectin of the present invention canhave an enhanced anti-inflammatory and/or an enhanced anti-fibrogeniceffects when compared to previous pectin compositions. In variousembodiments, the modified pectin of the present invention can inhibitsecretion of Tumor necrosis factor (TNF-α, cachexin), a protein involvedin systemic inflammation and a major member of a group of cytokines thatstimulate the acute phase reaction. It is produced chiefly by activatedmacrophages. In various embodiments, the modified pectin of the presentinvention does not cause cytotoxicity. In various embodiments, themodified pectin or modified pectin composition has potentanti-inflammatory and anti-fibrosis properties without exhibitingcytotoxicity. This represents a novel and unexpected combination ofeffects which suggest that the pharmaceutical-grade modified pectin canhave important effects in the treatment of human inflammatory diseases,fibrotic diseases, neoplastic diseases or combinations thereof.

In one aspect, methods for treating (e.g., controlling, relieving,ameliorating, alleviating, or slowing the progression of) or methods forpreventing (e.g., delaying the onset of or reducing the risk ofdeveloping) one or more diseases, disorders, or conditions in whichgalectins are involved, in a subject in need thereof are featured. Themethods include administering to the subject an effective amount of acompound of the invention, or a composition comprising the compound ofthe invention, to the subject.

As used herein, the term “effective dose” refers to the amount of acompound that, alone or in combination with an amount of a therapeuticagent, when administered as a parental, subcutaneous, inhaled,intra-articular, ocular, or oral formulation or to an animal or humanwith a galectin-dependent inflammatory, fibrotic or neoplastic diseaseresults in reduction in disease activity, as defined below in variousembodiments.

“Administration” refers to oral, or parenteral including intravenous,subcutaneous, topical, transdermal, intradermal, transmucosal,intraperitoneal, intramuscular, intracapsular, intraorbital,intracardiac, transtracheal, subcutaneous, subcuticular, intraarticular,subcapsular, subarachnoid, intraspinal, epidural and intrasternalinjection and infusion.

In some embodiments, the galactoarabino-rhamnogalacturonate compound hasterminal galactose residues that can constitute the main binding entityto galectin proteins. The remaining side chain composition may stabilizeor enhance its interaction with galectin proteins and thereby potentiatethe compound's therapeutic action. Interaction with galectins may not bethe only mechanism by which Compound G exerts its therapeutic action.While not wishing to be bound by speculation, the branchedgalactoarabino-rhamnogalacturonate compound of the invention caninteract with galectin proteins and can have wide therapeutic effectsthat have not been seen with galactose or small molecules derivatives ofgalactose.

In some embodiments, the compound of the instant invention is apolysaccharide chemically defined as galactoarabino-rhamnogalacturonate(also referred herein as “Compound G”), a branched heteropolymer havinga backbone characterized as a majority homopoly 1,4-linked galacturonicacid (GalA) molecules and methyl galacturonate (MeGalA) residues withintermittent short regions of alternating α-1,4-linked GalA andα-1,2-linked rhamnose (Rha), whereas the Rha units are further linked toside-chains. These side chains consist predominantly of1,4-β-D-galactose residues (Gal) and/or 1,5-α-L-arabinose residues(Ara). Other carbohydrates may be present in small amount on the Gal andAra side chains such as L-xylose (Xyl), D-glucose (Glu) and L-fucose(Fuc) residues. As used herein, the term “backbone” refers to the majorchain of a polysaccharide, or the chain originating from the major chainof a starting polysaccharide, having saccharide moieties sequentiallylinked by either alpha or beta glycosidic bonds. A backbone may compriseadditional monosaccharide moieties connected thereto at variouspositions along the sequential chain.

In some embodiments, Compound G is a chemically controlled modifiednatural branched heteropolymer of plant polysaccharides, some of whichare available commercially as USP pectin material. These natural pectinmaterials are of high average molecular weight ranging from 40,000 toover 1,000,000 Daltons (D) and are crude mixtures. Most common USPpectins are from citrus fruit, apple and beets, although others are alsoavailable.

In some embodiments, the Compound G composition can have an averagemolecular weight range of 2,000 to 80,000 D. In some embodiments, theaverage molecular weight of the compound G can range from 20,000 to70,000 D. In specific examples, the galactoarabino-rhamnogalacturonatemay have an average molecular weight of 5,000 to 55,000 D. Suchgalactoarabino-rhamnogalacturonate compounds can be obtained preferablythrough chemical and physical treatments and purification from naturalpectic substance of apple pectin. However compounds similar to CompoundG may be obtained from citrus, sugar beet pectin and other plants underappropriate extraction and chemical processing.

In one embodiment, the selectively depolymerizedgalactoarabino-rhamnogalacturonate (Compound G), a branchedheteropolymer, can have an average molecular weight of 2,000 to 80,000,or 20,000 to 70,000, or 5,000 to 55,000 Daltons, as determined by SEC-RIand MALLS method. As used herein, the term “depolymerization” refers topartial, selective or complete hydrolysis of the polysaccharide backboneoccurring, for example, when the polysaccharide is treated chemicallyresulting in fragments of reduced size when compared with the originalpolysaccharide.

Methods for Producing Compound G

Aspects of the invention relate to methods for producing selectivelydepolymerized galactoarabino-rhamnogalacturonate. In some embodiments,the process of the instant invention includes methods intended topreserve the structure and/or beta galactose characteristics andgalactose binding abilities of the side chains comprised primarily ofgalactose and arabinose, and to enhance the prevalence of galactosebinding moieties in the galactoarabino-rhamnogalacturonate compound. Insome embodiments, the methods can generate agalactoarabino-rhamnogalacturonate compound having a reduced averagemolecular weight, so as to be compatible with therapeutic formulationsfor pluralistic administration via routes, including but not limited to,intravenous, subcutaneous, inhaled, intra-articular, ocular, and oral.

In some embodiments, the compound can be synthesized from natural,highly branched, minimally processed and high methoxylated USP pectinwhich may come from any plant source, including but not limited to,citrus fruits, apple, or beet.

In some embodiments, the compound can be synthesized from natural,highly branched, minimally processed and high methoxylated USP pectinlike one manufactured from apple pomace containing 8-12% pectin.

In some embodiments, the compound can be synthesized under asufficiently controlled and specific hydrolysis of the glycosidic-linkedmethoxylated α-1,4-linked GalA while preserving the side-chains withenriched amounts of 1,4-β-D-Gal and 1,5-α-L-Ara. Amounts of 1,4-β-D-Galand 1,5-α-L-Ara can be quantitatively determined by GC-MS (Gaschromatography-mass spectroscopy) and AELC-PAD (anion exchange liquidchromatography-pulsed amperometric detector) methods.

In some embodiments, the compound can be produced by a processcomprising depolymerization catabolized by targeted peroxidationcleavage of glycosidic bonds (also known as beta elimination reaction)by ionized OH sup-generated from ascorbic acid and/or peroxide inpresence of a reduced form of a transition metal ion, like Cu sup.++.at1 to 100 mM. Other transition metals like Ca. sup.++ or Fe.sup.++ canalso be used for this purpose.

In some embodiments, the depolymerized compound can be exposed to pHrange of 8 to 10 for 10 to 30 minutes at temperature of 2 to 60° C. toinitiate controlled partial demethoxylation to generate a selectivelymiddle depolymerized compound with a degree of methoxylation of 40 to 70percent in comparison to initial levels of about 87%. The resultingcompound can be referred as middle-methoxylated compound. Completemethoxylation of galacturonic acid is considered to be approximately DE87%.

In some embodiments, the selectively-depolymerized polysaccharide of thepresent invention can have an endotoxin level of no more than 100 EU orno more than 300 EU as assessed by LAL method.

In some embodiments, the selectively-depolymerized polysaccharide of thepresent invention can have no more than 0.05% Nitrogenous impurities, asassessed by total nitrogen.

In some embodiments, the depolymerized composition can be exposed tomultiple washes of hot acidic alcohol (30-80° C.) to remove any residualendotoxin, copper and heavy metals, agricultural contaminates and otherimpurities.

Uses

In some embodiments, the therapeutic activity of the compound of theinvention can be derived from multiple beta-galactose and arabinosemoieties present on the compound. Such moieties can mimic cellularglycoproteins on cell surfaces and the extracellular matrix that bind togalectin proteins (e.g. galactose-binding proteins) which are highlyexpressed in inflammatory, fibrogenic, and tumorgenic processes. Theexpression of galectin-3 has been proven to be critical to inflammationand fibrogenesis in multiple organs, including but not limited to liver,kidney, heart, and lung. Modulating their activity may thus lead toinhibition and reversing of pathologic processes.

In another embodiment, the selectively-depolymerized polysaccharide ofthe present invention has no cytotoxicity and does not induce apoptosisin cell culture systems. As such, the selectively-depolymerizedpolysaccharide of the present invention differs from other knownpreparations of modified pectins that have been reported (See U.S. Pat.No. 8,128,966).

In another embodiment, the selectively-depolymerized polysaccharide ofthe present invention can have an anti-inflammatory effect on peripheralblood mononuclear cells (PMBC) and other inflammatory cell lines. Forexample, Compound G can reduce expression or response to inducers ofcytokine genes or proteins, including but not limited to, TNF-alpha. Theprocess of inflammation and repair can involve multiple cell typesincluding cells of the immune system and many inflammatory mediators ina complex and interconnected cascade of events. Acute inflammation canbe terminated or can progress to a chronic phase which may lead tofibrosis, a late stage of damage seen in a variety of human organdisease including but not limited to liver, lung, kidney, heart andpancreas.

Chronic inflammation in organs often leads to an accumulation offibrotic tissue. In fact, the end result of inflammation from multipleunderlying etiologies is generally fibrosis and resultant organdysfunction. This is evident in, for example, lung, heart, kidney,pancreas, and liver. Multiple lines of evidence point to galectinproteins, and galectin-3 in particular, as critical factors in thepathogenesis of organ fibrosis. It has, for example, been shown thatGalectin-3 knock out null mice are resistant to fibrosis of the liver inresponse to hepatotoxins, resistant to lung fibrosis in response tointra-tracheal bleomycin, and can be used as models of kidney fibrosis,heart fibrosis, and chronic pancreatitis (Henderson et al., “Theregulation of inflammation by galectin-3,” Immunol Rev. 230: 160-171,2009, lacobini et al., “Galectin-3 ablation protects mice fromdiet-induced NASH: a major scavenging role for galectin-3 in liver,” J.Hepatol. 54: 975-983, 2011, Lopez et al., “Gene expression profiling inlungs of chronic asthmatic mice treated with galectin-3: downregulationof inflammatory and regulatory genes,” Mediators Inflamm., 823279. Epub2011, Kolatsi-Joannou et al., “Modified citrus pectin reduces galectin-3expression and disease severity in experimental acute kidney injury,”PLoS One. 6(4): e18683, 2011).

“Fibrosis” refers to any tissue disorder, including, but not limited to,such cellular disorders as, for example, cirrhosis, kidney fibrosis,liver fibrosis, ovarian fibrosis, lung fibrosis, gastrointestinal orstomach fibrosis, and fibroids. The term “fibrosis” refers to both thepathological process leading from tissue injury through itsencapsulation by extracellular matrix, and the result of the process,which is a pathological formation of scar tissue.

“Cirrhosis” refers to any tissue disorder, including such cellulardisorders including, but not limited to, renal cirrhosis, livercirrhosis, ovarian cirrhosis, lung cirrhosis, gastrointestinal orstomach cirrhosis. The term “cirrhosis” refers to an advanced stage offibrosis, defined by the presence of encapsulated nodules. For purposesof this specification and claims, “cirrhosis” is considered to be a typeof fibrosis, and is included within the meaning of the term “fibrosis”used herein.

As used herein, “molecular markers”, “biochemical markers”,“biomarkers”, or “markers” are used interchangeably and refer toindividual molecules of biological origin, which can be monitored as a“readout” of specific metabolic events. These events are accompanied byformation of the “markers”, the quantitative level of which can often beused as an indication to advancement of the event.

Injury leading to fibrosis in the liver can occur in response to avariety of chronic insults, including but not limited to, alcohol abuse,drugs, toxins, fat deposition, viral hepatitis B and C, some metabolicdiseases causing chronic and/or permanent tissue irritation leading toinflammation and deposition of collagen, or fibrosis.

The advanced stage of liver fibrosis is cirrhosis, defined by thepresence of hepatocellular nodules encapsulated by broad bands offibrous tissue. Fibrosis is a systematic and coordinated response tochronic injury, developing through a series of highly coordinatedmolecular events, collectively called fibrogenesis. For example,fibrosis can develop as a result of chronic mammalian liver injury. Thesteps immediately following chronic liver injury can result in theactivation of hepatic stellate cells. The stellate cells' activation canlead to proliferation, fibrogenesis and cirrhosis. The activation eventsin stellate cells can be identified by specific molecular markers, suchas collagen I, alpha 1-smooth muscle actin, beta PDGF-receptor (aproliferation biomarker), matrix metalloproteinases and their inhibitorsMMP2, MMP9, TIMP1 and TMP2 (markers on matrix degradation), and avariety of cytokines, including but not limited to, TFG-beta1 (a markerof fibrogenesis). Development of fibrosis can be evaluated by thequantitative level of the respective markers. Reduction of fibrosis canbe evaluated by the decrease of the level of the respective markersduring various stages of fibrosis.

The pathophysiologic spectrum of fibrosis may be associated with serumbiomarkers including but not limited to hyaluronic acid and otherbreakdown products of collagens, cytokeratin-18 and other cytoskeletalcellular proteins, tissue inhibitor of metalloprotease I and II, otherliver derived collagen, matrix proteases or combinations thereof. Thesecompounds and/or biomarkers may be measured in serum or liver tissueusing immunoassays and the levels correlated with severity of diseaseand treatment.

The pathophysiologic spectrum of fibrosis also may be associated withserum biomarkers, including, but not limited to, reactive oxygenproducts of lipid or protein origin, lipid molecules or conjugates, orcombinations thereof. These biomarkers can be measured by various meansincluding immunoassays and electrophoresis and their levels correlatedwith severity of disease and treatment. Additional biomarkers mayinclude global shifts in proteomic analysis of serum or urine proteins.

The pathophysiologic spectrum of fibrosis also may be associated withserum biomarkers of NASH, a chronic metabolic inflammatory disorder thatleads to fibrosis and is described in this application. These biomarkerscan be cytokines, including but not limited to, TNF-alpha, TGF-beta orIL-8, or a metabolic profile of serum components that is indicative ofNASH presence or severity (these include serum and urine markers) orcombinations thereof. A profile of one or more of the cytokinesbiomarkers, as measured by immunoassay or proteomic assessment by LCmass spec, may provide an assessment of activity of the disease and amarker to follow in therapy of the disease.

The pathophysiologic spectrum of fibrosis in the liver also may beassociated with histopathological findings on liver biopsy, that includebut are not limited to, evidence of collagen deposition (including butnot limited to peri-sinusoidal, portal, central collagen deposition orcombinations thereof), portal to central bridging collagen deposition,hepatocellular nodules that distort the normal architecture,hepatocellular atypia consistent with malignant transformation orcombinations thereof.

The pathophysiologic spectrum of fibrosis in the liver may also beassociated with other pathological histological findings on liver biopsythat are associated with the underlying cause of chronic liver diseasethat results in fibrosis. Such findings can include, but are limited to,abnormalities in hepatocytes (including, but not limited to, ballooningdegeneration and intracellular hyaline and macrovesicular ormicrovesicular fat or combinations thereof), endothelial cells,macrophages, or bile duct cells and the infiltration of multiple typesof inflammatory cells, such as lymphocytes, monocytes, and/orneutrophils, or any combination of the foregoing.

The pathophysiologic spectrum of fibrosis can also includehistopathological findings on liver biopsy that are related to theunderlying disease of NASH. Such findings can include, but are notlimited to, evidence of intra-hepatocellular fat, hepatocellulartoxicity including but not limited to hyaline bodies, inflammatory cellinfiltrates (including but not limited to lymphocytes and varioussubsets of lymphocytes and neutrophils), changes in bile duct cells,changes in endothelial cells, number of Kupffer cell macrophages,collagen deposition (including but not limited to peri-sinusoidal,portal and central collagen deposition and portal to central bridgingcollagen deposition, hepatocellular nodules that distort the normalarchitecture, hepatocellular atypia consistent with malignanttransformation, and various scales and methods that combine various setsof observations for grading the severity of NASH or any combinations ofthe foregoing. Such histological assessments can be the sine-qua-none ofNASH diagnosis and therefore can integrally relate to assessment oftherapy.

The pathophysiologic spectrum of fibrosis can also includehistopathological findings on liver biopsy that examine the expressionof or change in expression of various molecules and their localizationin liver tissue or various cell types. Suitable molecules include, butare not limited to, various cytokine proteins. Cytokine proteins ofinterest can include, but are not limited to, TGF-beta, inflammatorymediators, reactive metabolite scavenger transport proteins, includingbut not limited to, CD36, and galectin proteins, including but notlimited to galectin-3 protein, or any combinations of the foregoing.

Clinical manifestations of fibrosis can include, but are not limited to,clinical testing of stage and severity of the disease, clinical signsand symptoms of disease, and/or medical complications resulting fromfibrosis. Clinical testing of stage and severity of liver fibrosis caninclude, but are not limited to, hematologic testing (including, but notlimited to, red blood cell count and/or morphology, white blood cellcount and/or differential and/or morphology, and/or platelet count andmorphology), serum or plasma lipids, including but not limited to,triglycerides, cholesterol, fatty acids, lipoprotein species and lipidperoxidation species, serum or plasma enzymes (including but not limitedto aspartate transaminase (AST), alanine transaminase (ALT), alkalinephosphatase (AP), gamma glutamyltranspeptidase (GGTP), lactatedehydrogenase (LDH) and isoforms, serum or plasma albumin and otherproteins indicative of liver synthetic capacity, serum or plasma levelsof bilirubin or other compounds indicative of the ability of the liverto clear metabolic byproducts, serum or plasma electrolytes (includingbut not limited to sodium, potassium, chloride, calcium, phosphorous),coagulation profile including but not limited to prothrombin time (PT),partial thromoplastin time (PTT), specific coagulation factor levels,bleeding time and platelet function. Clinical testing also includes butis not limited to non-invasive and invasive testing that assesses thearchitecture, structural integrity or function of the liver includingbut not limited to computerized tomography (CT scan), ultrasound (US),ultrasonic elastography (FibroScan) or other measurements of theelasticity of liver tissue, magnetic resonance scanning or spectroscopy,magnetic resonance elastography, percutaneous or skinny needle ortransjugular liver biopsy and histological assessment (including but notlimited to staining for different components using affinity dyes orimmunohistochemistry), measurement of hepatic portal-venous wedgepressure gradient, or other non-invasive or invasive tests that may bedeveloped for assessing severity of fibrosis in the liver tissue or anycombinations of the foregoing.

Clinical signs and symptoms of advanced fibrosis that has progressed tocirrhosis can include fatigue, muscle weight loss, spider angiomata,abdominal pain, abdominal swelling, ascites, gastrointestinal bleeding,other bleeding complications, easy bruising and ecchymoses, peripheraledema, hepatomegaly, nodular firm liver, somnolence, sleep disturbance,confusion, and/or coma. Medical complications of fibrosis are related tocirrhosis and include ascites, peripheral edema, esophageal and othergastrointestinal tract varices, gastrointestinal bleeding, otherbleeding complications, emaciation and muscle wasting, hepatorenalsyndrome, and hepatic encephalopathy. An additional complication offibrosis related cirrhosis is the development of complicationssufficiently severe to warrant placement on liver transplantation listor receiving a liver transplantation.

Nonalcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) arecommon liver disorders in the United States and Europe.Histopathologically, these disorders resemble alcoholic liver disease,but can occur in people who drink little or no alcohol. The pathologicalchanges in the liver include, but are not limited to, fat accumulationin hepatocytes, evidence of hepatocellular degeneration, infiltrates ofinflammatory cells, deposition of excess fibrous tissue, hepatocellularnodule formation, cirrhosis, hepatocellular carcinoma and combinationsthereof.

The major feature in NAFLD is fat accumulation in hepatocytes withminimal inflammation. NAFLD is usually identified on the basis of aliver biopsy performed because of mildly elevated liver transaminaselevels in the patient's serum or the suspicion of fatty liver onnon-invasive testing such as computerized tomography or ultrasound.

A subset of individuals with NAFLD are found to have NASH, which isfatty liver with the addition of the development of infiltration ofinflammatory cells (including but not limited to neutrophils orlymphocytes) within the lobule, central vein and portal areas andevidence of damage to hepatocytes, including but not limited to,ballooning degeneration. This inflammatory state of NASH may result inthe deposition of fibrous tissue, including but not limited to,collagen, which can lead to cirrhosis, nodule formation, and/orhepatocellular carcinoma.

The disease progress is insidious since most people with NASH feel welland are not aware that they have a liver problem. Despite the lack ofsymptoms, NASH can be severe and can lead to the deposition of fibroticmaterial in the liver which can result in severe scarring and/orcirrhosis and, in some cases, hepatocellular carcinoma.

The cause of liver injury in NASH is not known. Multiple theories havebeen proposed, with some experimental data to suggest their involvement.Some of these include, but are not limited to, hepatocyte resistance tothe action of insulin, production of inflammatory cytokines by fat cellsand other inflammatory cells that damage the liver and recruitadditional inflammatory cells and oxidative stress in hepatocytes withproduction of reactive oxygen radicals that damage liver cells andinduce inflammation.

To date no specific therapies for NASH or fibrosis exist and onlygeneral health recommendations are currently provided to patients. Theseinclude weight reduction, eating a balanced and healthy diet, increasingphysical activity, and avoidance of alcohol and unnecessary medications.Weight loss can improve serum liver tests in some patients with NASH andmay improve evidence of histological liver damage, but it does notreverse severe liver disease. In addition, it should be noted that notall patients with NASH are overweight.

A variety of experimental approaches have been evaluated, or are underevaluation in patients with NASH or fibrosis including, but not limitedto administration of antioxidants, such as vitamin E, selenium, betaine,and anti-diabetic agents including metformin, rosiglitazone, andpioglitazone. All clinical results to date have been disappointing. Insome embodiments, the compound of the present invention may be used inthe treatment of NASH or fibrosis. The compound of the present inventionmay be effective through manipulation of galectin proteins that areinvolved in pathogenesis and progression of diseases.

In some embodiments, the compound of the present invention (i.e.Compound G) has a clinically significant effect on fatty-livermetabolism in addition to fibrosis, whereas it may have a more of aclinically significant effect on related pathologies in addition toinflammation and fibrosis.

In some embodiments, the compound of the present invention has ananti-fibrotic effect in rat fibrosis model where fibrosis is induced bychemical toxin thioacetamide (TAA) which have similar pathology to theeffect of chronic consumption of alcohol that lead to fibrosis,cirrhosis and increase occurrence of hepatocarcinoma. (FIGS. 13-14)

In some embodiments, the compound of the present invention has ananti-inflammatory effect by reducing secretion of TNF alpha as depictedin an in-vitro model using PBMC cell stressed with microbial endotoxinto produce TNF alpha, a major cytokine, a biomarker and an inflammatoryprotein (FIG. 16).

Based on this discovery, the Compound G is proposed as therapy alone orin combination with other compounds listed above as treatment for humanNASH. In some embodiments, the compound of the present invention can beused for ameliorating or reversing hepatocyte fat accumulation,intra-portal and intra-lobular inflammatory infiltrate, and/or fibrosis,including but not limited to collagen deposition in the peri-sinusoidalspace, cirrhosis, and for preventing progression to hepatocellularcarcinoma.

In addition to NASH, there are multiple other chronic liver diseasesthat result in fibrosis and can progress to cirrhosis. For example,chronic liver diseases can include, but are not limited to, chronichepatitis virus infection (hepatitis B, C, and D), chronic alcoholabuse, biliary diseases (including, but not limited to, sclerosingcholangitis), primary biliary cirrhosis, genetic storage diseases, andmetal storage diseases (including but not limited to hemochromatosis andWilsons disease).

In some embodiments, Compound G may be effective in all chronic liverdiseases that lead to fibrosis regardless of the underlying etiology.

In some embodiments, Compound G may be effective in the treatment offibrosis in organs other than the liver, including, but not limited to,lung, kidney, heart and pancreas occurs through chronic inflammationleading to collagen deposition by cell types other than stellate cellswhich are specific to the liver. The cells responsible for fibrosis inother organs are myofibroblasts that have precursor cells that include,but are not limited to, resident tissue fibroblasts, circulatingfibrocytes, or epithelial cells generated through a process calledEMT—epithelial mesenchymal transformation.

Lung fibrosis can occur as a result of chronic inflammatory process in avariety of diseases including, but not limited to, idiopathic pulmonaryfibrosis, chronic obstructive lung disease, and chronic infections.

Fibrosis in the lung usually leads to stiffness of the lung tissuesresulting in reduced function of the lung, including but not limited to,reduced total lung capacity, vital capacity, forced expiratory volume,and diffusion capacity. These reduced functions can lead to clinicalsymptoms, including but not limited to, shortness of breath, reducedexercise tolerance, and reduced gas exchange resulting in low bloodoxygen levels. The ultimate result can be lung failure which requires alung transplant. There are no current pharmacological therapies for lungfibrosis.

Kidney fibrosis can occur as a result of multiple underlying diseases,including but not limited to, diabetes, obstruction of the urinarytract, hypertension, vascular disease, and autoimmune diseases.

Kidney fibrosis can progress to inhibit kidney function which results inreduced urine output and the accumulation of toxic metabolites in theblood stream. Kidney failure associated with kidney fibrosis requiresexternal support through dialysis or a kidney transplant. There are noknown current pharmacological therapies for kidney fibrosis.

Progressive heart failure is associated with multiple diseases,including but not limited to, chronic hypertension, coronary arterydisease, valvular heart disease, and hypertrophic heart disease. Heartfailure can occur in part because of the deposition of fibrous tissue inthe heart muscle, a process that has been shown to be associatedcausally with an increased expression of galectin-3. Progressive heartfailure can result in reduced contractility of the heart, dilation ofthe heart chambers, reduced cardiac output with multiple resultantsymptoms including edema, shortness of breath, reduced kidney function,mental confusion, and others. In the end stage, heart failure can onlybe treated with mechanical cardiac assist devices or hearttransplantation.

Chronic inflammation of the pancreas due most commonly, but notexclusively, to alcohol abuse, can result in fibrosis of the pancreasand reduced function of the exocrine and endocrine pancreas. Reducedexocrine function can lead to malabsorption of food and reducedendocrine function can lead to endocrine disorders, such as diabetes. Todate, there are no pharmacological therapies for pancreas fibrosis.

Galectin proteins have been shown to have multiple functions in cancercells including, but not limited to, enhancing invasiveness, causingresistance to chemotherapy, promoting metastasis, enhancingneovascularization, and allowing evasion of the immune system.

The vast majority of cancers express increased amounts of galectinproteins. Inhibition of galectins via administration of the compound ofthe present invention (e.g. Compound G) may be efficacious in therapy ofcancers that express galectins including but not limited cancers of theskin (squamous and melanoma), mouth, head and neck, lymphatic system,blood cells, alimentary tract (esophagus, stomach, small intestine,colon and rectum), pancreas, biliary tree, liver, lung, breast, kidney,ovary, testes, cervix, uterus, and neurological system

Aspects of the invention relate to a compound, or a compositioncomprising the compound, utilized for the treatment of inflammatory andfibrotic disorders in which galectins are involved in the pathogenesis,including but not limited to enhanced anti-fibrosis activity in organs,including but not limited to liver, kidney, lung, and heart. Otheraspects of the invention relate to the methods of treating inflammatoryand fibrotic disorders in which galectins are involved in thepathogenesis.

In some embodiments, the invention relates to a compound, a compositionthat has therapeutic activity or a method to reduce the pathology anddisease activity associated with nonalcoholic steatohepatitis (NASH)including, but not limited to, steatosis (fat accumulation inhepatocytes), ballooning degeneration of hepatocytes, inflammatoryinfiltrate in the liver, and deposition of collagen or fibrosis.

In some embodiments, the invention relates to a compound, or acomposition comprising the compound, utilized in treating inflammatoryand autoimmune disorders in which galectins are involved in thepathogenesis including, but not limited to, arthritis, rheumatoidarthritis, asthma, and inflammatory bowel disease (ulcerative colitisand Crohn's Disease).

In some embodiments, the invention relates to a compound, or acomposition comprising the compound, utilized in treating neoplasticconditions (e.g. cancers) in which galectins are involved in thepathogenesis by inhibiting processes promoted by the increase ingalectins, including, but not limited to, tumor cell invasion,metastasis, and neovascularization.

In some embodiments, the invention relates to a compound, or acomposition comprising the compound, utilized in enhancing or a methodfor enhancing the ability of tumor infiltrating T-cells, which areinhibited by the effect of tumor derived galectin proteins, to moreeffectively identify and kill tumor cells and thereby slow, stop orreverse the progression of tumors.

In some embodiments, the invention relates to a compound, a compositioncomprising the compound utilized in combination with tumor immunotherapywhich may be a vaccine directed towards specific tumor antigens oragents which activate or inhibit specific immune regulatory moleculesincluding but not limited to CTLA4, OX40, PD-1, or PD-L.

An effective dose of the compound of the present invention or acomposition comprising an effective dose of the compound can beadministered via a variety of routes including, parenteral via anintravenous infusion given as repeated bolus infusions or constantinfusion, intradermal injection, subcutaneously given as repeated bolusinjection or constant infusion, intra-articular injection, inhaled in anappropriate formulation, or oral administration.

The amount administered depends on the compound formulation, route ofadministration, etc. and is generally empirically determined in routinetrials, and variations will necessarily occur depending on the target,the host, and the route of administration, etc.

“Administration” refers to oral, or parenteral including intravenous,subcutaneous, topical, transdermal, intradermal, transmucosal,intraperitoneal, intramuscular, intracapsular, intraorbital,intracardiac, transtracheal, subcutaneous, subcuticular, intraarticular,subcapsular, subarachnoid, intraspinal, epidural and intrasternalinjection and infusion.

An effective parenteral dose of the compound of the present invention toan experimental animal can be within the range of 2 mg/kg up to 200mg/kg body weight given intravenously. An effective subcutaneousinjection dose of the compound of the present invention to an animal canbe within the range of 2 mg/kg up to 200 mg/kg body weight, or byintraperitoneal 2 mg/kg up to 200 mg/kg or by oral administration 10mg/kg or 50 mg/kg or 200 mg/kg or 1500 mg/kg body weight. Higher andlower doses can also be contemplated.

An effective parenteral dose of the compound of the present invention toa human subject can be within the range of 0.2 mg/kg up to 20 mg/kg bodyweight given intravenously. An effective subcutaneous injection dose ofthe compound of the present invention to a human subject can be in therange of 0.2 mg/kg up to 50 mg/kg body weight or by oral administration10 mg/kg up to 200 mg/kg body weight. Lower or higher doses than thoserecited above may be required. Specific dosage and treatment regimensfor any particular patient will depend upon a variety of factors,including the activity of the specific compound employed, the age, bodyweight, general health status, sex, diet, time of administration, rateof excretion, drug combination, the severity and course of the disease,condition or symptoms, the patient's disposition to the disease,condition or symptoms, and the judgment of the treating physician.

An effective parental dose may be given daily (in one or divided doses),three times weekly, two times weekly, or monthly via intravenous,intradermal, subcutaneous or other routes as practiced by the medicalprofessional to administrate drugs.

An effective oral dose of the compound of the present invention to anexperimental animal or human may be formulated with a variety ofexcipients and additives that enhance the absorption of the compound viathe stomach and small intestine.

An effective oral dose could be 10 times and up to 100 times the amountof the effective parental dose.

An effective oral dose may be given daily, in one or divided doses ortwice, three times weekly, or monthly.

The compound of the present invention or compositions comprising thecompound of the present invention may be administered orally; or byintravenous injection; or by injection directly into an affected tissue,as for example by injection into an arthritic joint. In some instancesthe compound or composition may be administered topically, as in theform of eye drops, nasal sprays, ointments or the like. Also, othertechniques such as transdermal delivery systems, inhalation or the likemay be employed.

In some embodiments, the compounds described herein can beco-administered with one or more other therapeutic agents. In certainembodiments, the additional agents may be administered separately, aspart of a multiple dose regimen, from the compounds of this invention(e.g., sequentially, e.g., on different overlapping schedules with theadministration of the compound of the invention. In other embodiments,these agents may be part of a single dosage form, mixed together withthe compounds of this invention in a single composition. In stillanother embodiment, these agents can be given as a separate dose that isadministered at about the same time that the compound of the invention.When the compositions include a combination of the compound of thisinvention and one or more additional therapeutic or prophylactic agents,both the compound and the additional agent can be present at dosagelevels of between about 1 to 100%, and more preferably between about 5to 95% of the dosage normally administered in a monotherapy regimen.

In some embodiments, a therapeutically effective amount of thedepolymerized compound or of the composition can be compatible andeffective in combination with a therapeutically effective amount ofvarious anti-oxidant compounds (e.g. glycyrrhizin, ascorbic acid,L-glutathione, cysteamine etc) as described in U.S. Pat. No 7,078,064.

An effective dose given to an animal or human subject with NASH andliver fibrosis means the amount of a compound that, alone or incombination with amount of other therapeutic agent, when administered asa parental, subcutaneous, inhaled, intra-articular, ocular, or oralformulation results in at least a 10% reduction in hepatocellular fat,hepatocytes with ballooning degeneration, inflammatory cell infiltrated,at least a one point reduction in the NAFLD activity score, or at leasta 10% reduction in collagen deposition in the liver assessed byhistological staining with Sirius red, or slowing the progression ofdeposition of fibrotic tissue in the liver by at least 10%.

An effective dose given to a human subject with NASH and liver fibrosiscan result in at least a 10% reduction in serum biomarkers associatedwith NASH, or at least a 10% improvement in hepatocyte fat content orliver stiffness as assessed by ultrasound or MR elastography, or atleast a 10% improvement in liver function tests that measure metabolicfunction or shunting in the liver, or at least a 10% reduction inclinical symptoms and complications resulting from liver fibrosis andcirrhosis including but not limited to symptoms and complicationsresulting from reduced metabolic and elimination processes (includingbut not limited to bilirubin), reduced liver synthetic capacity(including but not limited to albumin and coagulation proteins), portalhypertension, and hepatic encephalopathy.

An effective dose given to a human subject with NASH and liver fibrosismeans an improvement in clinical parameters or reduced progression ofclinical parameters when compared to a control untreated group of humansubjects including but not limited to at least a 10% reduction in serumbiomarkers associated with NASH, or at least a 10% improvement inhepatocyte fat content or liver stiffness as assessed by ultrasound orMR elastography, or at least a 10% improvement in liver function teststhat measure metabolic function or shunting in the liver, or at least a10% reduction in clinical symptoms and complications resulting fromliver fibrosis and cirrhosis including but not limited to symptoms andcomplications resulting from reduced metabolic and elimination processes(including but not limited to bilirubin), reduced liver syntheticcapacity (including but not limited to albumin and coagulationproteins), portal hypertension, and hepatic encephalopathy.

An effective dose given to an animal or human subject with liverfibrosis or cirrhosis due to a disorder other than NASH means the amountof a compound that, alone or in combination with amount of othertherapeutic agent, when administered as a parental, subcutaneous,inhaled, intra-articular, ocular, or oral formulation results in, forexample, at least a 10% reduction in collagen deposition in the liverassessed by histological staining with Sirius red, or slowing theprogression of deposition of fibrotic tissue in the liver by at least10%.

An effective dose given to a human subject with liver fibrosis orcirrhosis due to a disorder other than NASH means the amount of acompound that, alone or in combination with amount of other therapeuticagent, when administered as a parental, subcutaneous, inhaled,intra-articular, ocular, or oral formulation results in, but is notlimited to, at least a 10% reduction in serum biomarkers associated withliver fibrosis, or at least a 10% improvement in liver stiffness asassessed by ultrasound or MR elastography, or at least a 10% improvementin liver function tests that measure metabolic function or shunting inthe liver, or at least a 10% reduction in clinical symptoms andcomplications resulting from liver fibrosis and cirrhosis including butnot limited to symptoms and complications resulting from reducedmetabolic and elimination processes (including but not limited tobilirubin), reduced liver synthetic capacity (including but not limitedto albumin and coagulation proteins), portal hypertension, and hepaticencephalopathy.

An effective dose given to a human subject with liver fibrosis orcirrhosis due to a disorder other than NASH means an improvement inclinical parameters or reduced progression of clinical parameters whencompared to a control untreated group of human subjects including butnot limited to at least a 10% reduction in serum biomarkers associatedwith fibrosis, or at least a 10% improvement in liver stiffness asassessed by ultrasound or MR elastography, or at least a 10% improvementin liver function tests that measure metabolic function or shunting inthe liver, or at least a 10% reduction in clinical symptoms andcomplications resulting from liver fibrosis and cirrhosis including butnot limited to symptoms and complications resulting from reducedmetabolic and elimination processes (including but not limited tobilirubin), reduced liver synthetic capacity (including but not limitedto albumin and coagulation proteins), portal hypertension, and hepaticencephalopathy.

An effective dose given to an animal or human subject with kidneyfibrosis means an improvement in clinical parameters or reducedprogression of clinical parameters when compared to a control untreatedgroup of animals or human subjects including but not limited to at leasta 10% reduction in kidney fibrosis assessed by histology, or a least a10% improvement in proteinuria, or at least a 10% improvement inglomerular filtration rate, or at least a 10% improvement in clinicalsigns and symptoms related to renal insufficiency.

An effective dose given to an animal or human subject with lung fibrosismeans an improvement in clinical parameters or reduced progression ofclinical parameters when compared to a control untreated group ofanimals or human subjects including but not limited to at least a 10%reduction in lung fibrotic tissue assessed on histology, 10% improvementin lung volumes, or at least a 10% improvement in expiratory volumes, orat least a 10% improvement in clinical signs and symptoms related topulmonary insufficiency.

An effective dose given to an animal or human subject with heartfibrosis means an improvement in clinical parameters or reducedprogression of clinical parameters when compared to a control untreatedgroup of animals or human subjects including but not limited to at leasta 10% reduction in heart fibrotic tissue assessed on histology, 10%improvement in heart contractility, or at least a 10% improvement incardiac output, or at least a 10% improvement in clinical signs andsymptoms related to heart failure.

An effective dose given to an animal or human subject with pancreaticfibrosis means an improvement in clinical parameters or reducedprogression of clinical parameters when compared to a control untreatedgroup of animals or human subjects including but not limited to at leasta 10% reduction in pancreas fibrotic tissue assessed on histology, 5%improvement in synthesis or secretion of pancreatic exocrine enzymes, orat least a 5% improvement in pancreatic endocrine enzymes including butnot limited to insulin, or at least a 10% improvement in clinical signsand symptoms related to pancreatic insufficiency.

An effective dose given to an animal or human subject with cancer,either as a single agent or in combination with other cancerchemotherapy, immunotherapy, or tumor vaccines, means an improvement inclinical parameters or reduced progression of clinical parameters whencompared to a control untreated group of animals or human subjectsincluding but not limited to at least a 10% reduction in size of tumors,or at least a 10% reduction in number of cancer metastases, or at leasta 10% increase in activity of immune system against the cancer cells, orat least a 10% improvement in clinical signs and symptoms related tocancer including but not limited to improvement in progression freesurvival, or overall survival, or reduced adverse effects of therapy, orimproved quality of life.

While a number of embodiments of the present invention have beendescribed, it is understood that these embodiments are illustrativeonly, and not restrictive, and that many modifications and/oralternative embodiments may become apparent to those of ordinary skillin the art. For example, any steps may be performed in any desired order(and any desired steps may be added and/or any desired steps may bedeleted). Therefore, it will be understood that the appended claims areintended to cover all such modifications and embodiments that comewithin the spirit and scope of the present invention.

The invention will be further described in the following examples. Itshould be understood that these examples are for illustrative purposesonly and are not to be construed as limiting this invention in anymanner.

EXAMPLES

For reference, the selectively depolymerized product of the instantinvention (Compound G) refers to a compound prepared in accordance withExample 1 described herein, Compound D refers to a referencepolysaccharide prepared in accordance with the methods disclosed in U.S.Pat. No. 7,893,252, incorporated herein by reference in its entirety.Compound H refers to a high molecular depolymerized pectinpolysaccharide (90,000 to 140,000 D), one not prepared in accordancewith Example 1 described herein, but rather prepared according with themethods disclosed in U.S. Pat. No. 8,236,780, incorporated herein byreference in its entirety. Compound S refers to a polysaccharidemanufactured from USP Citrus pectin according to the methods disclosedin U.S. Pat. No. 8,128,966. The invention provides manufacturing processof modified pectin, and use in cancer treatment. And it is incorporatedherein by reference in its entirety. Compound T refers to a commercialMCP “Thorne Research Fractionated Pectin Powder 5 OZ” purchased onAmazon.com.

Example 1: Manufacturing of Compound G

The selectively depolymerized product of the instant invention wasprepared by a process illustrated in FIG. 1.

Apple pectin USP HM (50 kg) was dissolved and heated in water to 35-85°C. 1 M HCl or NaOH was added in order to pH-adjust the solution to pH5-7 and mixed well. The mixing was continued for 2 hours at the 35-85°C. set-point. 1M NaOH or HCl was added as needed to readjust pH tobetween 5 and 7. Solution was cooled to 30° C. At 30° C., pH wasadjusted to between 5 and 7.

CuSO₄ is added to the pH-adjusted pectin solution so as to result in afinal 1 mM CuSO₄ concentration. The 1 mM CuSO₄ solution was mixed for 30minutes at a temperature of between 10° C. and 30° C.

At the conclusion of the 30 minutes, 1 mM CuSO₄ mixing step, 50 gramssodium ascorbate was added (amount was pre-calibrated to achieve thedesired MW) and mixed for 5 to 20 minutes. H₂O₂ was added start with0.02 and up to 1.0 moles/kg pectin (pre-calibrated for initial startingpectin MW) and the H₂O₂ concentration was maintained for 4 hours (usingquantitative test, Sigma, St-Louis) while the solution pH was maintainedbetween 4 and 7.

5M NaOH was added to the solution so as to result in a solution pH ofbetween 8 and 10. The pH-adjusted solution was mixed for 10-30 minutes.Concentrated HCL was then added to the pH-adjusted solution to adjustthe pH of the solution to between 4 and 5. The solution, once adjustedto pH between 4 and 5 can be kept mixing for 2 to 24 hours between 2° C.and 8° C.

Solution was then heated to 80° C. for 30-180 minutes and 1-5 kg ofFilter-Aid was added (Celite) to the solution, and the solution withadded Celite was stirred for 30 minutes and then filtered. The solidsresulting from the filtration were discarded.

The filtrate was concentrated 1.5-3× under vacuum, and then pH-adjustedto between 3 and 5. Hot ethanol or isopropanol was added on a 50%weight. The mixture was stirred 1-2 hours to precipitate product, andthe mixture was then filtered. The filtrate was discarded, leaving awhite to off-white precipitate.

Cold 96% EtOH was added to the solution and the resulting slurry wasthen stirred for 30 minutes. The solution was filtered and the filtratewas discarded. The 96% EtOH slurry step was repeated, followed by afinal filtration and recovery of a white to off-white precipitate.

The final product of this process yields a composition with general themolecular structure shown in FIG. 2, as assessed by the analysesdescribed in Examples 2, 3, 4, and 5 below.

Example 2: Analysis of Average Molecular Weight by MALLS

A Multi-Angle Laser Light Scattering detection system can be used togenerate a ZIMM plot that independently predicts the molecular weight ofpolymers. The principle of the MALLS method is based on the fact thatlight is more strongly scattered by large molecules than by smallmolecules. The output of the light scattering detector is proportionalto the multiplication of the concentration and the average molecularweight of macromolecules. Therefore, the shape of the light scatteringpeak is asymmetric. Molecular weight versus the elution volume isobtained and average molecular weights and average molecular weightdistributions can be calculated.

Table 1 below and FIGS. 3A-3B illustrate a determination of the averagemolecular weight of a selectively depolymerized compound of the instantinvention by MALLS, indicating an average molecular weight ofapproximately 37 kilodaltons (kD) in EDTA buffer solution with astandard deviation of 8%.

TABLE 1 Study Number Sample ID Results (Daltons) 3098-003 3098-003-00001(EDTA) 36.950 [STD 8%]

Example 3: Analysis by High Performance Liquid Chromatography (HPLC)

Size Exclusion Chromatography (SEC) is a well-established techniqueusing HPLC for the characterization of polymers. SEC in combination withRefractive Index (RI) detection is used for the determination of averagemolecular weights of polymeric carbohydrates by retention time profile.FIG. 4a demonstrates the elution time of standard polysaccharides andFIG. 4b shows the molecular weight profile of Compound G.

From this analysis and as shown in FIG. 4b , range of molecular weightof Compound G is from 20 to 70 kDa.

Example 4: Determination of Glycosyl composition of Compound G

Glycosyl composition analysis was performed by combined gaschromatography followed by mass spectrometry (GC/MS) of theper-O-trimethylsilyl (TMS) derivatives of the monosaccharide methylglycosides produced from the sample by acidic methanolysis.

An aliquot of each sample was taken and added to tube with 20 ug ofinositol as the internal standard. Methyl glycosides were then preparedfrom the dry sample following methanolysis in 3 M HCl in methanol at 80°C. (6 hours), followed by re-N-acetylation with pyridine and aceticanhydride in methanol (for detection of amino sugars). The sample wasthen per-O-trimethylsilylated by treatment with Tri-Sil (Pierce) at 80°C. (0.3 hours). These procedures were carried out as previouslydescribed in Merkle and Poppe (1994) Methods Enzymol. 230: 1-15; York,et al. (1985) Methods Enzymol. 118:3-40. GC/MS analysis of the TMSmethyl glycosides was performed on an AT 6890N GC interfaced to a 5975BMSD, using a Supelco EC-1 fused silica capillary column (30 m×0.25 mmID).

The results of the composition analysis are listed in the table below:

TABLE 2 Mol % of monosaccharide residues of Compound G by GC-MS SampleGlycosyl residue Mass (g)* Mol %¹ Comp G Arabinose (Ara)  13.4  5.6 Lot# Rhamnose (Rha)  9.0  3.4 S126K4208 Fucose (Fuc) n.d. — Xylose (Xyl) 5.2  2.2 Glucuronic Acid (GlcA) n.d. — Galacturonic acid (GalA) 220.871.7 Mannose (Man) n.d. — Galactose (Gal)  45.4 15.9 Glucose (Glc)  3.3 1.2 N-AcetylGalactosamine n.d. — (GalNAc) N-AcetylGlucosamine n.d. —(GlcNAc) N-AcetylMannosamine n.d. — (ManNAc) Total 297.1 CHO = 99%*Assay standard deviation has not been determined

Example 5: Determination of Glycosidic Linkages and Structure by H1 andC13 NMR

NMR spectroscopy reveals individual molecular signatures and linkages,providing a type of analytical fingerprint for complex carbohydratemolecules. Two-dimensional NMR spectra were evaluated to reveal themolecular fingerprint of the composition of the instant invention,Compound G, and comparison to Compound S.

For NMR spectroscopy, the samples of Compound G and Compound S weredissolved in 0.7 mL D20 (99.96% D), and transferred to a 5-mm NMR tube(Wilmad). 1-D Proton and 2-D TOCSY, NOESY, gradient enhanced COSY(gCOSY), HSQC, and gHMBC NMR spectra were acquired on a Varian Inova-500MHz spectrometer at 343 K (70° C.). Chemical shifts were measuredrelative to internal acetone (oH=2.225 ppm, oC=31 .07 ppm).

Table 3 below indicates approximate ratios of methyl galacturonate togalacturonic acid, and of methyl galacturonate and galacturonic acid togalactose, obtained from 2-D HSQC NMR of Compound G:

TABLE 3 Compound G SD* GalA-6-OMe/GalA 1:2 GalA(-6-OMe)/Gal 2:1

The major components in Compound G are 4-linked galacturonic acid,4-linked methyl galacturonate, and 4-linked galactose. Rhamnose isclearly present in the HSQC spectrum. Table 4 contains the NMR spectralassignment of Compound G.

TABLE 4 Position Residue 1 2 3 4 5 6 4-α-GalAp ¹H 5.08 3.77 3.98 4.424.69 ¹³C 100.0 69.5 69.9 79.3 72.3 175.9 4-α-GalApOMe^(a) ¹H 4.92 3.753.99 4.47 5.10 ¹³C 100.8 69.5 69.9 79.3 71.5 172.1 4-β-Galp ¹H 4.62 3.693.77 4.16 3.72 3.81/3.13 ¹³C 105.5 73.1 74.2 78.5 75.4 61.7 t-α-Araf ¹H5.17 4.13 3.97 4.06 3.83/373 ¹³C 107.8 82.0 77.4 84.8 62.1 5-α-Araf ¹H5.08 4.14 4.02 4.21 3.88/3.80 ¹³C 108.6 82.0 77.7 83.2 67.6 Rhap ¹H n.d.n.d. 3.89 3.40 3.79 1.25 ¹³C n.d. n.d. n.d. n.d. n.d. 17.6 ^(a)methylresonance: 3.81/53.9 ppm

FIG. 5a shows the two-dimensional NMR spectrum for Compound G and FIG.5b shows the two-dimensional NMR spectrum for Compound S.

Comparison of two-dimensional NMR spectra from different samples ofmodified pectin material is a powerful method for evaluating differentstructures.

FIG. 5c shows the overlapping spectra of Compound G and Compound S,revealing the marked differences in the two-dimensional NMR spectra ofthese two compounds. The positions that are circled are in the spectrumof Compound G, but not in Compound S. This indicates importantstructural differences between Compound G and Compound S.

This analysis demonstrates clear structural differences between CompoundG and Compound S, made by a different manufacturing method. Thesestructural differences are evident on two-dimensional NMR analyses whenthe chemical composition analysis, which measures only themonosaccharide composition, does not show gross differences.

Two-dimensional NMR fingerprinting can be included in the certificate ofanalysis for GMP batches of Compound G as a precise evaluation of thecomplex molecular structure.

Example 6: Determination of Compound G Cytotoxicity to Cultured CellLines

One of the prominent features of modified pectin compounds that havebeen reported (U.S. 8,128,966 and U.S. 2012/0149658) is theircytotoxicity in cell lines of various types, including the induction ofapoptosis. For example, U.S. Pat. No. 8,128,966 discloses modifiedpectins that induce apoptosis in cancer cells, such as B16-F10 melanomacell line.

Unlike the results of U.S. Pat. No. 8,128,966, the modified pectin ofthe present invention, was shown to have no cytotoxicity when used in avariety of cell lines, including B16-F10 melanoma cells.

Human hepatic stellate cell line, LX-2 is routinely used as a tool foranalysis of hepatic fibrosis. The LX-2 cells proliferate normally in 2%FC serum rich media. However, once stress in 0.1% FC serum, LX-2 cellsgo through similar pathological changes as established in fibroticliver. Cytotoxicity was assessed using Compound D, Compound H, andCompound G.

FIG. 6a shows the results of a proliferation assay utilizing theincorporation of tritium-labeled thymidine into growing LX-2 cells.There was no difference from control cells in thymidine incorporation at48 hours of culture when cells were incubated with 1 mg/ml of CompoundD, Compound H, or Compound G.

FIG. 6b shows the results of a cell viability assay utilizing a vitaldye which is taken up by non-viable cells and excluded from viablecells. There was no difference from control cells in cell viability at48 hours of culture when cells were incubated with 1 mg/ml of CompoundD, Compound H, or Compound G.

The presence of apoptosis was also evaluated using the annexin apoptosiskit (eBioscience). Annexins are a family of calcium-dependentphospholipid binding proteins that preferentially bindphosphatidylserine (PS). Under normal physiologic conditions, PS ispredominantly located in the inner leaflet of the plasma membrane. Uponinitiation of apoptosis, PS loses is asymmetric distribution across thephospholipid bilayer and is translocated to the extracellular membraneleaflet marking cells as targets of phagocytosis. Once on the outersurface of the membrane, PS can be detected fluorescently labeledAnnexin V in a calcium-dependent manner.

FIG. 6c shows the results of FACS (Fluorescence activated cell sorting)analysis of LX-2 cells to assess apoptosis which showed no evidence ofapoptosis in cells treated with Compound D or Compound G versus controlcells (vehicle treated).

As a further test for apoptosis, LX-2 cells were examined for thepresence of DNA fragmentation. FIG. 6d shows that there was no evidenceof DNA fragmentation in cells treated with Compound D (D1 or D2),Compound H or Compound G as compared to control.

Macrophages are integrally involved in inflammatory and fibroticprocesses. Thus, a macrophage cell line THP-1 (ATCC® Number: TIB-202TM)was evaluated for the effect of Compound G on cytotoxicity as shown inFIG. 7. The THP-1 cell line is a monocyte type cells line harvested fromperipheral blood of patient (Homo sapiens) with Acute Monocytic Leukemia(AML) (see Tsuchiya S, et al. Induction of maturation in cultured humanmonocytic leukemia cells by a phorbol diester. Cancer Res. 42:1530-1536, 1982).

The THP-1 cells were suspended in assay media containing 10%FBS. About25,300 cells/well were transferred at 100 ul/well to 96 well plates. At24 hours the culture media was changed to fresh media and the cells wereincubated over night. Test compounds were serially diluted in assaymedia containing 10% FBS and transfer 100 ul/well to the growingmonocyte cells THP-1. The final assay volume was 200 ul/well containing10% FBS, 2× Gentamicin, and the following test articles: Compound G ofthe present invention and Compound D, a galactomannan product or inpresence of digitoxin. The cells were incubated for 3 days with the testarticles. After removing 50 ul of supernatant for other testing,cytotoxicity/growth was measured by adding 15 ul of Promega “CellTiter96® Aqueous One Solution” to the 96 wells and viability was monitored atOD 490 nm after 1-7.5 hours. The CellTiter 96® AQueous One Solution CellProliferation Assay is a colorimetric method for determining the numberof viable cells in proliferation, cytotoxicity or chemosensitivityassays. The CellTiter 96® AQueous One Solution Reagent contains atetrazolium compound[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt; MTS] and an electron coupling reagent (phenazineethosulfate; PES). PES has enhanced chemical stability, which allows itto be combined with MTS to form a stable solution for detection cellviability in in-vitro culture media.

FIG. 7 shows that increasing amounts of Compound G applied to THP-1cells did not result in cytotoxicity.

The melanoma cell line B16-F10 was previously used as an assay foractivity of modified pectins. Thus, B16-F10 cells were evaluated for theeffect of Compound G on cytotoxicity.

The melanoma cell line B16-F10 (ATCC® Number: CRL-6475™) are a mixtureof spindle-shaped and epithelial-like cells from skin melanoma of mouse(Mus musculus, Strain: C57BL/6J (See Fidler I J. Biological behavior ofmalignant melanoma cells correlated to their survival in vivo. CancerRes. 35: 218-224, 1975.)

Melanoma cells B16-F10 were transferred to a fresh media (DMEM—10% FetalBovine Serum—FBS). About 2,900 cells/well (passage #4) were transferredin 100 ul/well to 96 well plates for overnight incubation. At 24 hoursthe culture media was changed to fresh serum free media and incubatedover night. Test compounds were serially diluted in assay mediacontaining 1% FBS and 100 ul/well was transferred to the growingmelanoma cells. The final assay volume was 200 ul/well containing 1%FBS, 2× Gentamicin, and compound G of the present invention, compound D,a galactomannan product or 5-fluorouracil. The cells were incubated for3 days with the test compounds. Cytotoxicity/growth was measured byadding 20 ul of Promega “CellTiter 96® Aqueous One Solution” to the 96wells and monitored at OD 490 nm after 1 hour. The CellTiter 96® AQueousOne Solution Cell Proliferation Assay is a colorimetric method fordetermining the number of viable cells in proliferation, cytotoxicity orchemosensitivity assays. The CellTiter 96® AQueous One Solution Reagentcontains a tetrazolium compound[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt; MTS] and an electron coupling reagent (phenazineethosulfate; PES). PES has enhanced chemical stability, which allows itto be combined with MTS to form a stable solution for detection cellviability in in-vitro culture media.

FIG. 8 shows that increasing amounts of Compound G applied to B16-F10cells did not result in cytotoxicity. This represents a clear differencefrom the function of other modified pectin compounds previouslydescribed.

PBMC cells are primary cell extracted from whole blood using ficoll, ahydrophilic polysaccharide. Peripheral blood mononuclear cells (PBMC)are integrally involved in inflammatory and fibrotic processes. Thus,PBMC were evaluated for the effect of Compound G on cytotoxicity.

FIG. 9 shows that Compound G from three different lots applied to PBMCsdid not result in cytotoxicity. Compound G at concentrations of up to500 ug/mL had no effect on the growth of PBMC. Cytotoxicity was measuredusing MTS assay (CellTiter 96® AQueous One Solution Cell ProliferationAssay (MTS) Promega USA.)

FIG. 10 shows that increasing amounts of Compound G applied to culturedlung fibroblast cells did not result in cytotoxicity. The MRC-5 cellline is a normal fibroblast that was derived from normal lung tissue(Homo sapiens (human) male (ATCC Catalog No. CCL-171™). Increasingamounts of the following test articles: Compound G of the presentinvention, Compound D, a galactomannan product was tested using aCellTiter 96® AQueous One Solution Cell Proliferation Assay(MTS)—Promega USA. FIG. 10 shows that Compound G at up to 800 ug/mL hadno effect on the growth of lung fibroblast in vitro.

The summary of these data show convincingly that Compound G is notcytotoxic to cells and does not induce apoptosis. This is in contrast toall the other reports of modified pectin materials reported.

Example 7: In-Vitro Assay for Anti-Inflammatory Effect Using PBMCPrimary Cells

Compound G was assessed for its biological activity in inflammatoryconditions. Multiple cells are involved in and activated in theinflammatory process. One key cell type in this process are PeripheralBlood Mononuclear Cells (PMBCs), a primary cell routinely used asanti-inflammatory in-vitro model, which are activated by inflammatorymediators, recruited to sites of inflammation, transform into tissuemacrophages, and enhance the inflammatory process. Therefore, PBMCs wereused as an in vitro model to evaluate the effect of Compound G.

The in-vitro assay for inflammation was developed by stressing PBMC withmicrobial endotoxin (microbial lipopolysaccharides) and measuring bysecretion of TNF alpha, a major cytokine and biomarker for inflammation.PBMC were re-suspended in assay media containing 10% FBS (Gibco lot#749413), 2× Gentamicin, and L-Glutamine. PMBC were transferred 90μl/well to assay plate (169,000 cells/well). 90 μl/well of assay mediawas added to assay plate to a total volume of 180 μl/well for approx. 16hours. LPS (microbial toxin) was serially diluted in assay media. 20μl/well of LPS were added to assay plate to a final volume of 200μl/well and incubated for 7 hours at 37 degrees C.60 μl/well wereremoved for TNF-α (Transforming Nuclear Factor alpha) ELISA assay. The60 μl samples were diluted 1:4 with ELISA diluent (total volume is 240μl) and 100 μl/well sample were transferred to an ELISA plate. h-TNF-αwas analyzed using an ELISA Development Kit (PeproTech, cat #900-K25,lot #0509025), with Human TNF-α as standard on an ELISA plate.100 μl ofABTS Liquid Substrate was added to each well and OD was read at 405 nmwith a wavelength correction set at 650 nm.

The plate plan used is set out below:

Plate Plan: 1 2 3 4 5 8 7 8 9 10 11 12 A h- LPS; 400 ng/ml (1:2) h- BTNFa TNFa C STD; Compound S; 1.0 mg/ml (1:2); Assay Media Containing 50STD; D 5 ng/ml ng/ml LPS 5 ng/ml E (1:2) Compound D; 1.0 mg/ml (1:2);Assay Media Containing 50 (1:2) F ng/ml LPS G Compound G; 1.0 mg/ml(1:2); Assay Media Containing 50 H ng/ml LPS

FIG. 11 demonstrates graphically results of Plate 364 with h-TNF alfaELISA. Compound G at 0.5 mg/mL reduced 50% of TNF-alpha secretion bystressed PBMC primary cells. The reduction of TNF-alpha secretion issignificantly higher than either Compound D or Compound S referencepolysaccharides tested.

The ability of Compound D, Compound H, and Compound G to inhibitTNF-alpha secretion in PBMC were directly compared, as shown in FIG. 12.Compound H was produced using the method disclosed in U.S. Pat. No.8,236,780. Compound D and Compound H did not show any ability to reduceTNF-alpha secretion from PBMC cells, in fact Compound H appearedincrease TNF-alpha. In contrast, Compound G significantly inhibitedsecretion of TNF-alpha.

Compound G was shown to be a potent inhibitor of an in vitro model ofinflammation. Such activity was shown to be absent from Compound S orCompound H, modified pectin compounds made through different processesfrom Compound G. Therefore, results form Examples 6 and 7 showed thatCompound G is modified pectin having unique non-cytotoxic andanti-inflammatory properties that were not described for other knownpectin-derived compounds.

Example 8: In-Vitro Induction of Fibrogenesis in Liver LX-2 StellateCell Line

In other experiments, the inventors have shown that human culturedstellate cell lines (LX-2) express and secrete into the mediumgalectin-1 and galectin-3 upon stressing the cells by reducing the FCserum in the culturing media from 2% to 0.1%.

Human hepatic stellate cell line, LX-2 is routinely used as a tool foranalysis of hepatic fibrosis. The LX-2 cells proliferate normally in 2%FC serum rich media. However, once stress in 0.1% FC serum they gothrough similar pathological changes as established in fibrotic liver

The Compound G may also modulate, increase or decrease in LX-2 stellatecells molecules or biomarkers that are involved in fibrogenesis,including but not limited to collagen I, II, Ill, IV, metalloproteases,inhibitors of metalloproteases, and cytokines.

The Compound G compound may also modulate expression of cytokines andlipids and reactive oxygen species in liver macrophages, or Kupffercells.

The Compound G compound may also modulate the expression ofhepatocellular genes, uptake and metabolism of lipids and reactiveoxygen species.

The effect of Compound G on the expression of galectin-3 in both intraand extra-cellular compartments was assessed, as shown in FIG. 13. Whilerich serum media had produced negligible amount of galectin-3, when LX-2cells (human stellate cell line) were stressed with growth mediadepleted of fetal calf serum (in only 0.1% serum media), a model whichhave been shown to be an in-vitro model for fibrogenesis, galectin-3 wasexpressed reaching maximum at about 5 to 7 days post culture in 0.1%serum media. An immunochemistry staining technique demonstratedincreased expression of galectin-3 at day 6 post-stress in 0.1% serum,while the addition of compound G was shown to significantly suppress theexpression of galectin-3.

Example 11: Assessment of Therapy in a TAA-Induced Liver Fibrosis Model

In order to determine if the compound of the instant invention has ananti-fibrotic biological activity in living animals, a preliminaryfeasibility test was conducted in vivo. Severe liver fibrosis wasinduced by chemical toxicity of biweekly administration of thioacetamide(TAA). Compound G was given IP at 90 mg/kg weekly for 4 weeks, andcompound D was given at dose of 180 mg/kg weekly for 4 weeks. Theexperimental design is shown in FIG. 14.

After 8 weeks of treatment with TAA, Sirius red staining of the fibroticliver (vehicle control) showed extensive infiltration of fibrotic tissue(FIG. 15). In contrast, the fibrotic material was markedly reduced, andnearly eliminated in some areas in the liver from an animal treated withCompound G (FIG. 15).

Statistical analysis of fibrosis grade and percent collagen wasperformed with animals treated with Compound G and Compound D (FIG. 16).

FIG. 16 shows a statistically significant reduction in fibrotic areameasured by digital morphometric analysis of Sirius red-stained liversections when animals were treated with Compound G and Compound D whencompared to vehicle controls, with greater reduction seen with CompoundG when compared to Compound D.

These experiments demonstrate anti-fibrotic activity of Compound G,which correlates with its anti-inflammatory effect demonstrated in celllines.

Example 12: Assessment of Therapy in Mouse Fatty-Liver NASH Model

The effect of galectin binding carbohydrates in the therapy ofexperimental fatty liver disease and NASH was examined. STAM mice inwhich diabetes was induced and a high fat diet was administered wereused as an experimental model. This is a proven model in which the miceconsistently develop NASH with hepatocyte fat accumulation, evidence ofhepatocyte toxicity, portal and lobular inflammatory infiltrates,peri-sinusoidal fibrosis, advanced fibrosis with nodule formation,cirrhosis, and ultimately hepatocellular carcinoma in a certainpercentage of animals. NASH mice were treated biweekly IV at 9-12 weeksof study, as shown in FIG. 17.

STAM mice were used to explore the effect of Compound G. In the studytwo compounds have been tested, Compound D (as described in U.S. Pat.No. 7,893,252) and Compound G on the histopathological findingsassociated with NASH in the liver (FIG. 17). In the STAM model, neonatalmice were given an injection of streptozotocin which results inendocrine pancreatic insufficiency and diabetes mellitus. At four weeksof age, a high fat diet was introduced which was continued throughoutthe experiment. This model results in a reproducible disease thatincludes fatty liver (FL), NASH, NASH with fibrosis (Fib), noduleformation (N) and in a certain percentage of animals hepatocellularcarcinoma (HC). In this experimental design, drug therapy was initiatedat 8 weeks and continued for a total of 4 weeks. Compound D wasadministered at a dose of 120 mg/kg dissolved in normal salineintravenously twice a week. Compound G was administered in a dose of 60mg/kg dissolved in normal saline intravenously twice a week.

FIG. 18 shows that mice in all groups gained weight over the time of theexperiment with no differences between groups. This result indicates ata gross level that there was little toxic effect of the treatments onthe animals and any changes detected are unlikely due to the overallhealth of the animals. Overall there were 2 deaths in the vehicle group(2/12, 17%) and one in the Compound D treated group (1/12, 8%), allrelated to liver disease as determined by postmortem examination by theveterinarian. There were no deaths in the Compound G treated group. Thisin vivo activity of lack of toxicity in the mice correlates with thelack of cytotoxicity seen in vitro cell line experiments.

The NAFLD activity score was used to evaluate disease severity and givespoints for three aspects of NASH pathology including, steatosis (0(<5%), 1 (5-33%), 2 (33-66%), or 3 (>66%)), hepatocyte ballooning (0(none), 1, (few), or 3 (many)), and lobular inflammation (0 (no foci), 1(<2 foci/200× field), 2 (2-4 foci/200× field), or 3 (>4 foci/200×field)). The total number of points is the NAFLD activity score.

FIG. 19A and FIG. 19B show a graphical depiction with statistical valuesof the NAFLD activity score in the three experimental groups. There wasan improvement in NAFLD activity score in animals treated with CompoundG and less of an effect with Compound D.

FIG. 20A and FIG. 20B show a graphical depiction with statistical valuesof the percent collagen in the three experimental groups. Sirius red isa histological stain that has a specific affinity for collagen fibers,staining them red, and is therefore a quantitative tool for assessingthe degree of fibrosis in liver biopsies. The area of Sirius redstaining on liver histopathological sections from each of the threetreatment groups was assessed using computer assisted morphometricanalysis. Animals in both the early and late treatment groups had amarked reduction in collagen proportional area when treated withCompound G. Treatment with Compound D had an intermediate effect oncollagen proportional area between vehicle control and Compound G. Theseresults demonstrated that treatment with Compound G significantlyreduces liver fibrosis in mice with NASH.

The results in the NASH mice (FIGS. 17-20) extend and confirm theresults found in the TAA treated rats on liver fibrosis. Additionally,these experiments show a marked anti-inflammatory effect of Compound Gwith a reduction in the NAFLD activity score (steatosis, hepatocyteballooning, and inflammatory infiltrate). The combination of theseanti-inflammatory and anti-fibrotic effects in an animal model of NASHcorrelates with the anti-inflammatory effect demonstrated in the PBMCcell culture model.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications of changesin light thereof are to be included within the spirit and purview ofthis application and scope of the appended claims. All publications,patents and patent applications cited herein are hereby incorporated byreference in their entirety for all purposes.

What is claimed is:
 1. An arabinogalacto-rhamnogalacturonan compoundcomprising a 1,4-linked galacturonic acid (GalA) and methylgalacturonate (MeGalA) residues backbone linked to branchedheteropolymers of alternating oligomers of α-1,2 linked rhamnose andα-1,4-linked GalA residues, the rhamnose residues carrying a primarybranching of oligomers of 1,4-β-D-galactose residues, 1,5-α-L-arabinoseresidues, or combinations thereof, wherein thearabinogalacto-rhamnogalacturonan compound is capable of reducing thesecretion of TNF-alpha cytokine from monocytes stressed with endotoxin.2. The compound of claim 1, wherein the compound is capable of reducingthe secretion of TNF alpha by activated monocytes/macrophages by atleast 25%.
 3. The compound of claim 1, wherein the 1,4-linkedgalacturonic acid and methyl galacturonate residues backbone representsbetween 55 to 85 molar percent of the total carbohydrate molar content,the branched heteropolymer of alternating α-1,2 linked rhamnose andα-1,4-linked GalA residues represents between 1 and 6 molar percent ofthe total carbohydrate molar content, the oligomer 1,4-β-D-galactose ofthe primary branching represents between 6 to 15 molar percent of thetotal carbohydrate molar content and the oligomer 1,5-α-L-arabinose ofthe primary branching represents between 2 to 8 molar percent of thetotal carbohydrate molar content, as characterized by gaschromatography/mass spectrometry.
 4. The compound of claim 1, furthercomprising xylose, glucose, fucose residues or combination thereof. 5.The compound of claim 1, wherein the oligomer of 1,4-β-D-galactoseresidues, 1,5-α-L-arabinose residues or combinations thereof representsat least 8 molar percent of the total carbohydrate molar content.
 6. Thecompound of claim 1, wherein the compound has a 1,4-β-D-galactose to1,5-α-L-arabinose residues ratio ranging from 2:1 to a 3:1.
 7. Thecompound of claim 1, wherein the compound has an average molecularweight ranging from 2 kDa to 80 kDa.
 8. The compound of claim 1, whereinthe compound has an average molecular weight ranging from 20 kDa to 70kDa.
 9. The compound of claim 1, wherein the compound has an averagemolecular weight ranging from 5 kDa to 55 kDa.
 10. The compound of claim1, wherein the compound has a degree of methoxylation ranging from 40%to 70% of the maximum of 87%.
 11. The compound of claim 1, wherein thecompound has a methyl galacturonate to galacturonic acid ratio rangingfrom 2:1 to 1:2.
 12. The compound of claim 1, wherein the compound has amethyl galacturonate plus galacturonic acid ratio to galactose rangingfrom 4:1 to 7:1
 13. The compound of claim 1, wherein the compound issubstantially free of microbial endotoxin, agricultural pesticides,agricultural herbicides, copper, heavy metals, proteins, nitrogenouscompounds or any combination of the foregoing.
 14. The compound of claim1, wherein the compound does not induce decreased viability when used totreat LX2 immortalized human hepatic stellate cells.
 15. The compound ofclaim 1, wherein the compound is capable of reducing expression ofgalectin-3 or a substantial decrease in secretion of galectin-3 whenused to treat stressed LX2 immortalized human hepatic stellate cellsproducing galectin-3.
 16. A composition comprising a compound accordingto claim 1 in an acceptable pharmaceutical carrier, for use intherapeutic formulations.
 17. The composition of claim 16, wherein thecomposition is administered parenterally via an intravenous,subcutaneous, or oral route.
 18. The composition of claim 16, furthercomprising a therapeutic agent.
 19. The composition of claim 16, whereinthe therapeutic agent is an anti-oxidant compound, an anti-inflammatoryagent, vitamins, an immunotherapeutic agent, a neutraceutical supplementor combinations thereof.
 20. The composition of claim 16, for use in thetreatment of nonalcoholic steatohepatitis, fibrosis, inflammatorydisorder, autoimmune disorders, neoplastic conditions or of cancer.